Vsig8-based chimeric proteins

ABSTRACT

The present invention relates, in part, to, chimeric proteins which include the extracellular domain of V-set and immunoglobulin domain-containing protein 8 (VSIG8) and their use in the treatment of diseases, such as immunotherapies for cancer and/or inflammatory diseases.

PRIORITY

This application is a continuation of U.S. application Ser. No.16/484,854, filed Aug. 9, 2019, which is a 371 National Stage entry ofPCT/US18/20038, filed Feb. 27, 2018, which claims the benefit of, andpriority to, U.S. Provisional Application No. 62/463,999, filed Feb. 27,2017, the contents of which are hereby incorporated by reference intheir entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“SHK-003C2_Sequence_Listing-ST25”. The sequence listing is 81,795 bytesin size, and was created on or about Nov. 1, 2021. The sequence listingis hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates, in part, to, chimeric proteins whichinclude the extracellular domain of V-set and immunoglobulindomain-containing protein 8 (VSIG8) and their use in the treatment ofdiseases, such as immunotherapies for cancer and/or inflammatorydiseases.

BACKGROUND

Recent clinical data have demonstrated impressive patient responses toagents targeting immune coinhibitory molecules, including, for example,clinical trials that led to the approval of YERVOY, KEYTRUDA, andOPDIVO. These immunotherapies are collectively characterized ascheckpoint inhibitors, and unfortunately, these therapies only provideclinical benefit for ˜15-30% of cancer patients. One potential approachto improving clinical response rates for a broader population of cancerpatients includes combining a checkpoint inhibitor therapeutic withanother therapy. Such combinations, when applied using multipleindividual therapeutics, might lead to improved clinical benefit but arecumbersome to develop. Further, many immunotherapies are complicated bysevere side effects that significantly narrow a patient's therapeuticwindow for treatment.

There remains a need for novel methods and compositions that provideeffective immunotherapies, including consolidating multiple therapeuticmechanisms into single drugs.

SUMMARY

Accordingly, the present invention provides, in part, compositions andmethods that find use in cancer treatment by, for instance, overcomingmultiple suppressive mechanisms, in the tumor microenvironment, andstimulating immune antitumor mechanisms. Similarly, the compositions andmethods find use in treating an inflammatory disease.

In embodiments, the present chimeric protein masks immune inhibitorysignals and/or enhances immune stimulatory signals in a singleconstruct. In embodiments, such immune modulating effects are achievedthrough direct receptor/ligand interactions.

For instance, the present invention provides, in part, compositions andmethods that allow for contemporaneously inhibiting VISTA/VISIG8signaling and stimulating OX40/OX40L signaling in antigen-presentingcells. Such concurrent VISIG8 blockade and OX40 agonism causes, interalia, an overall decrease in immunosuppressive cells and a shift towarda more inflammatory milieu and an increased antitumor effect.

In aspects, the present invention provides a heterologous chimericprotein comprising: (a) a first domain comprising a portion of V-set andimmunoglobulin domain-containing protein 8 (VSIG8) that is capable ofbinding a VSIG8 ligand; (b) a second domain comprising a portion of OX40Ligand (OX40L) that is capable of binding a OX40L receptor; and (c) alinker linking the first domain and the second domain. In aspects, thepresent invention provides methods of treating cancer with thisheterologous chimeric protein. In aspects, the present inventionprovides methods of treating an inflammatory disease with thisheterologous chimeric protein.

In aspects, the present invention provides a recombinant fusion proteincomprising a general structure of: N terminus—(a)—(b)—(c)—C terminus,where (a) is a first domain comprising an extracellular domain of VSIG8that is at least 95% identical to the amino acid sequence of SEQ ID NO:2 and is capable of binding a VSIG8 ligand, (b) is a linker linking thefirst domain and the second domain and comprising a hinge-CH2-CH3 Fcdomain derived from human IgG4 (e.g. 95% identical to the amino acidsequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27), and (c) isa second domain comprising an extracellular domain of OX40 ligand(OX40L) that is at least 95% identical to the amino acid sequence of SEQID NO: 4 and is capable of binding an OX40L receptor. In embodiments,the present invention provides methods of treating cancer with thisheterologous chimeric protein. In embodiments, the present inventionprovides methods of treating an inflammatory disease with thisheterologous chimeric protein.

In embodiments, the present chimeric proteins are capable of, or finduse in methods involving, shifting the balance of immune cells in favorof immune attack of a tumor or any other unwanted cells. For instance,the present chimeric proteins can shift the ratio of immune cells at asite of clinical importance in favor of cells that can kill a tumor(e.g. T cells, cytotoxic T lymphocytes, T helper cells, natural killer(NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g.M1 macrophages), B cells, and dendritic cells and in opposition to cellsthat protect tumors (e.g. myeloid-derived suppressor cells (MDSCs),regulatory T cells (Tregs); tumor associated neutrophils (TANs), M2macrophages, and tumor associated macrophages (TAMs)). In embodiments,the chimeric protein enhances the recognition of tumor antigens by CD8+T cells and/or enhances tumor infiltration by these T cells.

In aspects, the present chimeric protein and/or recombinant fusionprotein is used in a method for treating cancer or an inflammatorydisease comprising administering an effective amount of a pharmaceuticalcomposition comprising the chimeric protein to a patient in needthereof. In cancer treatment embodiments, for example, the presentchimeric protein and/or recombinant fusion protein generates an immunememory response.

Aspects include uses of the present chimeric protein and/or recombinantfusion protein in the manufacture of a medicament, e.g., for treating acancer and/or an inflammatory disease.

Any aspect or embodiment described herein can be combined with any otheraspect or embodiment as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows, without wishing to be bound by theory, an in silicopredicted secondary structure of a human VSIG8-Fc-OX40L chimericprotein, with each of its three domains in its predicted natural state.The predicted molecular weight of the chimeric protein is approximately68.1 kDa. FIG. 1B shows a synapse that has formed by a chimeric proteinbetween a tumor cell and a T cell.

FIG. 2 shows characterization of human VSIG8-Fc-OX40L chimeric proteinby Western blot analysis under non-reducing, reducing, andreducing/deglycosylated (PNGase) conditions. Specifically, eachindividual domain of the chimeric construct was probed using ananti-VSIG8, anti-Fc, or anti-OX40L antibody. Untreated samples of thehVSIG8-Fc-OX40L chimeric protein, e.g. control, were loaded into lane 2in all the blots (no β-mercaptoethanol or PNGase). Samples in lane 3 ofall the blots were treated with the reducing agent, β-mercaptoethanol,while samples in lane 4 of all the blots were treated with the amidasePeptide:N-Glycosidase enzyme. The band sizes confirm the predictedmonomeric molecular weight of approximately 68.1 kDa and suggest thatthe chimeric protein's native state is as a glycosylated dimer

FIG. 3 show graphs of functional ELISAs demonstrating binding of humanVSIG8-Fc-OX40L to human IgG (left panel) and to recombinant OX40 (rightpanel).

FIG. 4 is a graph showing in vitro cell binding of human VSIG8-Fc-OX40Lto OX40-expressing cell lines (i.e., Jurkat cells engineered tooverexpress human OX40). Binding was performed in vitro and analyzed byflow cytometry.

FIG. 5 is a graph showing octet binding affinities for humanVSIG8-Fc-OX40L. The binding affinity of VSIG8-Fc-OX40L to OX40 wasmeasured by biolayer interferometry (Octet), as compared tocommercially-available single-sided human Fc-OX40L control or totavolixumab (an anti-human OX40 antibody). The data indicate thatVSIG8-Fc-OX40L binds to human OX40 with high affinity, measured at 767pM (top curve: tavolixumab, middle curve: VSIG8-Fc-OX40L, bottom curve:single-sided human Fc-OX40L).

FIG. 6 is a graph showing in vitro NF-κB/NIK signaling assay using thehuman VSIG8-Fc-OX40L chimeric protein. U2OS cells from the DiscoverX NIKsignaling assay were cultured with 30 μg/mL of either acommercially-available single-sided Fc-OX40L, an anti-OX40 antibody, ananti-OX40L antibody, or the hVSIG8-Fc-OX40L chimeric protein. Bars leftto right are: single-sided Fc-OX40L, an anti-OX40L antibody, ananti-OX40 antibody, and hVSIG8-Fc-OX40L chimeric protein.

FIG. 7 is graph showing a Staphylococcus enterotoxin B (SEB)super-antigen activation/cytokine release assay. Human PBMCs werecultured with Staphylococcal enterotoxin B (200 ng/mL) +/− titrations ofcommercially-available single-sided human Fc-OX40L,commercially-available single-sided VSIG8-Fc, a combination of the twosingle-sided molecules, or the human VSIG8-Fc-OX40L chimeric protein.Three days later, supernatants were assessed using ELISAs specific tohuman IL2.

FIG. 8 show characterization of murine VSIG8-Fc-OX40L chimeric proteinby Western blot analysis under non-reducing, reducing, andreducing/deglycosylated (PNGase) conditions. The band sizes confirm thepredicted monomeric molecular weight of approximately 68.1 kDa andsuggest that the chimeric protein's native state is as a glycosylateddimer.

FIG. 9A to FIG. 9C show ELISA assays demonstrating binding affinity ofthe different domains of murine VSIG8-Fc-OX40L chimeric protein fortheir respective binding partners. FIG. 9A shows the binding anddetection of mVSIG8-Fc-OX40L chimeric protein to VISTA, the bindingpartner for VSIG8. A commercially-available mVSIG8-Fc standard isunavailable; therefore, no standard curve was generated. FIG. 9B showsthe binding and detection of the Fc portion of the mVSIG8-Fc-OX40Lchimeric protein to plate-bound anti-human IgG antibodies. Mouse Ig(mIg) was used as a standard (circle symbols). Detection was via an HRPconjugated anti-human IgG antibody. It was observed that in ELISA assaysgenerally, using the central Fc region to detect chimeric proteinstended to underestimate the actual protein content in a sample.Therefore, low levels of the VSIG8-Fc-OX40L chimeric protein (squaresymbols) was detected compared to standard (circle symbols). FIG. 9Cshows the binding and detection of mVSIG8-Fc-OX40L chimeric protein toplate-bound recombinant murine OX40, a binding partner for OX40L anddetecting via an OX40L-specific antibody. Murine OX40L was used as astandard (circle symbols).

FIG. 10A and FIG. 10B are graphs showing in vitro cell binding assays ofmurine VSIG8-Fc-OX40L to a VISTA-expressing cell line and to anOX40-expressing cell lines. Immortalized cell lines were engineered tostably express (FIG. 10A) murine VISTA (EL4-mVISTA) or (FIG. 10B) murineOX40 (CHOK1-mOX40). Increasing concentrations of mVSIG8-Fc-OX40L wasincubated with each parental and over-expressing cell line for twohours. Cells were collected, washed, and stained with antibodies fordetection of chimeric protein binding by flow cytometry. All engineeredcell lines bound mVSIG8-Fc-OX40L in a concentration-dependent manner atlow nM in vitro cell binding affinities; mVSIG8-Fc-OX40L to CHOK1/mOX40at 16 nM and mVSIG8-Fc-OX40L to EL4/mVISTA at 56 nM.

FIG. 11 is a graph showing an Octet-based assessment of binding affinityof murine VSIG8-Fc-OX40L to murine OX40-His. 10 μg/mL of eithercommercially-available mFc-OX40L (bottom curve) or the mVSIG8-Fc-OX40Lchimeric protein (top curve) were bound to penta-his biosensors coatedwith recombinant murine OX40-his.

FIG. 12 is a graph showing an in vitro NF-κB-luciferase signaling assayusing the murine VSIG8-Fc-OX40L chimeric protein. Jurkat cellsengineered to express NF-κB-luciferase and OX40, were cultured with 18nM of either an irrelevant protein (the negative control, left bar), acommercially-available single-sided Fc-OX40L (middle bar), or themVSIG8-Fc-OX40L (right bar).

FIG. 13A to FIG. 13C shows immunophenotyping from tumor bearing micetreated with murine VSIG8-Fc-OX40L. Mice were inoculated with CT26tumors on day 0. Once the tumors were palpable and at least 4 to 6 mm indiameter, mice were treated with two doses of 150 μg of themVSIG8-Fc-OX40L chimeric protein. Immunophenotyping was performed on asubset of mice from each treatment group on various tissues collected onday 13 after tumor implantation. This data demonstrate that mice treatedwith the mVSIG8-Fc-OX40L chimeric protein exhibited higher percentagesof total CD4+ T cells in the spleen, tumor-draining lymph node (TDLN),and tumor (FIG. 13A), and this increase was comprised of a majorityincrease in CD4+ CD25− T cells (FIG. 13B). Further analysis wasperformed by tetramer staining to analyze the fraction of CD8+ T cellsthat recognize the AH1 tumor antigen natively expressed by CT26 tumors.Within both the spleen and tumor, a higher proportion of those T cellswere found to recognize the AH1 tumor antigen in mice treated withmVSIG8-Fc-OX40L, as compared to other groups (FIG. 13C). For each panelof FIG. 13A to FIG. 13C the conditions are untreated or treated withmurine VSIG8-Fc-OX40L.

FIG. 14A to FIG. 14C show anti-tumor efficacy of murine VSIG8-Fc-OX40Lagainst colorectal CT26 tumor. Balb/c mice were inoculated with CT26tumors on day 0. Following four days of tumor growth, when tumorsreached a diameter of 4 to 5 mm, mice were treated with either controlantibodies or the mVSIG8-Fc-OX40L chimeric protein. Treatments were thenrepeated again on day 7. FIG. 14A shows individual tumor growth curvesfor each treatment group, FIG. 14B shows overall survival through day 50of the experiment, and FIG. 14C is a table summarizing the group sizesand treatment outcomes for each group.

FIG. 15 is a graph showing an ELISA-based anti-drug antibody (ADA) assayof the murine VSIG8-Fc-OX40L chimeric protein.

FIG. 16 shows four potential configurations of illustrative chimericproteins (PD1-Fc-OX40L).

FIG. 17 shows Western blots of PD1-Fc-OX40L chimeric proteins run onSDS-PAGE under a non-reducing condition, a reducing condition, and areducing condition and following treatment with Peptide-N-Glycosidase F(PNGaseF).

FIG. 18 shows a chromatograph for PD1-Fc-OX40L chimeric proteins run onSize Exclusion Chromatography (SEC).

FIG. 19 shows SDS-PAGE and native (non-SDS) PAGE gels for PD1-Fc-OX40Lchimeric proteins run under a non-reducing condition (“−”) or under areducing condition (“+”).

FIG. 20 shows a native (non-SDS) PAGE gel for PD1-No Fc-OX40L chimericproteins which lack an Fc domain in a linker.

FIG. 21 shows, without wishing to be bound by theory, a model for how ahexamer and concatemers form from chimeric proteins of the presentinvention.

FIG. 22 is a table showing joining linkers and Fc linkers that can becombined into exemplary modular linkers. The exemplary modular linkersshown can be combined with any herein-described Type I and Type IIproteins and/or extracellular domains of a herein described Type I andType II proteins to form a chimeric protein of the present invention.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery of engineeredchimeric proteins comprising the extracellular domain of V-set andimmunoglobulin domain-containing protein 8 (VSIG8) near theamino-terminus. In embodiments, the chimeric protein further comprisesthe extracellular domain of OX40 ligand (OX40L) near thecarboxy-terminus. In embodiments, the two extracellular domains areconnected by a linker. In embodiments, the present chimeric proteinmasks an immune inhibitory signal on tumor cells replacing it with animmune stimulatory signal for the effective treatment of cancers.

Chimeric Proteins

In embodiments, the present invention relates to chimeric proteinsengineered to comprise the extracellular domain of the immune inhibitoryreceptor V-set and immunoglobulin domain-containing protein 8 (VSIG8).VSIG8 is a single-pass type I membrane protein which functions as areceptor for V-region Immunoglobulin-containing Suppressor of T cellActivation (VISTA). Specifically, the human VSIG8 protein comprises 414amino acids including a 21 amino acid signal sequence, a 242 amino acidextracellular domain (ECD) containing 2 Ig-like V-type(immunoglobulin-like) domains, a 21 amino acid transmembrane domain, anda 130 amino acid cytoplasmic domain.

In embodiments, the present chimeric protein comprises a domain, e.g.,the extracellular domain, of human VSIG8. The human VSIG8 comprises theamino acid sequence of SEQ ID NO: 1 (with the amino acid sequence of theextracellular domain comprising SEQ ID NO: 2).

In embodiments, the present chimeric proteins may comprise theextracellular domain of VSIG8 as described herein (e.g., SEQ ID NO: 2),or a variant or a functional fragment thereof. For instance, thechimeric protein may comprise a sequence of the extracellular domain ofVSIG8 as provided above, or a variant or functional fragment thereofhaving at least about 60%, or at least about 61%, or at least about 62%,or at least about 63%, or at least about 64%, or at least about 65%, orat least about 66%, or at least about 67%, or at least about 68%, or atleast about 69%, or at least about 70%, or at least about 71%, or atleast about 72%, or at least about 73%, or at least about 74%, or atleast about 75%, or at least about 76%, or at least about 77%, or atleast about 78%, or at least about 79%, or at least about 80%, or atleast about 81%, or at least about 82%, or at least about 83%, or atleast about 84%, or at least about 85%, or at least about 86%, or atleast about 87%, or at least about 88%, or at least about 89%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99% sequence identity with the amino acid sequence of theextracellular domain of VSIG8 as described herein.

In embodiments, the present chimeric proteins may comprise a variantextracellular domain of VSIG8 in which the signal peptide (e.g., asprovided in SEQ ID NO:1) is replaced with an alternative signal peptide.In embodiments, the present chimeric protein may comprise a variantextracellular domain of VSIG8 which is expressed from a cDNA that hasbeen codon-optimized for expression in protein producing cells such asChinese Hamster Ovary (CHO) or Human Embryonic Kidney (HEK) cells.

In embodiments, an extracellular domain of VSIG8 refers to a portion ofthe protein which is capable of interacting with the extracellularenvironment. In embodiments, the extracellular domain of VSIG8 is theentire amino acid sequence of the protein which is external of a cell orthe cell membrane. In embodiments, the extracellular domain of VSIG8 isa portion of an amino acid sequence of the protein which is external ofa cell or the cell membrane and is needed for signal transduction and/orligand binding as may be assayed using methods known in the art (e.g. invitro ligand binding and/or cellular activation assays).

In embodiments, the extracellular domain of VSIG8 refers to a portion ofthe protein which is capable for binding to V-regionImmunoglobulin-containing Suppressor of T-cell Activation (VISTA). VISTAis a negative checkpoint regulator that is involved with suppressing theactivation of resting T cells including CD4+ or CD8+ T cells. Binding ofVSIG8 with VISTA induces a suppressive effect on T cell activation,proliferation and/or immune cytokine production. In embodiments, thechimeric protein binds to human VISTA with a K_(D) of less than about 1μM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 90 nM,about 80 nM, about 70 nM, about 60 nM, about 55 nM, about 50 nM, about45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM,about 15 nM, about 10 nM, or about 5 nM, or about 1 nM (as measured, forexample, by surface plasmon resonance or biolayer interferometry). Inembodiments, the chimeric protein binds to human VISTA with a K_(D) ofless than about 1 nM, about 900 pM, about 800 pM, about 700 pM, about600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM about 55 pMabout 50 pM about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25pM, about 20 pM, about 15 pM, or about 10 pM, or about 1 pM (asmeasured, for example, by surface plasmon resonance or biolayerinterferometry). In embodiments, the chimeric protein binds to humanVISTA with a K_(D) of from about 200 pM to about 600 pM (as measured,for example, by surface plasmon resonance or biolayer interferometry).

In embodiments, the present chimeric protein further comprises a domain,e.g., the extracellular domain, of the immune stimulatory molecule OX40ligand (OX40L). OX40L is a type II transmembrane glycoprotein belongingto the Tumor Necrosis Factor (TNF) superfamily. Specifically, the humanOX40L protein comprises 183 amino acids including an amino-terminalcytoplasmic domain (amino acids 1-23) and a carboxy-terminalextracellular domain (amino acids 51-183).

In embodiments, the present chimeric protein comprises the extracellulardomain of human OX40L. The human OX40L comprises the amino acid sequenceof SEQ ID NO: 3 (with the amino acid sequence of the extracellulardomain comprising SEQ ID NO: 4).

In embodiments, the present chimeric proteins may comprise theextracellular domain of OX40L as described herein (e.g., SEQ ID NO: 4),or a variant or functional fragment thereof. For instance, the chimericprotein may comprise a sequence of the extracellular domain of OX40L asprovided above, or a variant or functional fragment thereof having atleast about 60%, or at least about 61%, or at least about 62%, or atleast about 63%, or at least about 64%, or at least about 65%, or atleast about 66%, or at least about 67%, or at least about 68%, or atleast about 69%, or at least about 70%, or at least about 71%, or atleast about 72%, or at least about 73%, or at least about 74%, or atleast about 75%, or at least about 76%, or at least about 77%, or atleast about 78%, or at least about 79%, or at least about 80%, or atleast about 81%, or at least about 82%, or at least about 83%, or atleast about 84%, or at least about 85%, or at least about 86%, or atleast about 87%, or at least about 88%, or at least about 89%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99%) sequence identity with the amino acid sequence of theextracellular domain of OX40L as described herein.

In embodiments, the present chimeric proteins may comprise a variantextracellular domain of OX40L in which the signal peptide (e.g., asprovided in SEQ ID NO: 3) is replaced with an alternative signalpeptide. In embodiments, the present chimeric protein may comprise avariant extracellular domain of OX40L which is expressed from a cDNAthat has been codon-optimized for expression in protein producing cellssuch as CHO or HEK cells.

In embodiments, the extracellular domain of OX40L refers to a portion ofprotein which is capable of interacting with the extracellularenvironment. In embodiments, the extracellular domain of OX40L is theentire amino acid sequence of the protein which is external of a cell orthe cell membrane. In embodiments, the extracellular domain of OX40L isa portion of an amino acid sequence of the protein which is external ofa cell or the cell membrane and is needed for signal transduction and/orligand binding as may be assayed using methods know in the art.

In embodiments, the extracellular domain of OX40L refers to a portion ofthe protein which is capable for binding to the OX40 receptor. Similarto other TNF superfamily members, membrane-bound OX40L exists as ahomotrimer. OX40L binds to OX40, a member of the TNF receptorsuperfamily that is expressed predominantly on CD4+ and/or CD8+ T cellsas well as a number of lymphoid and non-lymphoid cells. Evidencesuggests that the major function of the OX40-OX40L interaction is totransmit a late co-stimulatory signal to promote the survival andproliferation of activated T cells and prolong immune responses.

In embodiments, the chimeric protein of the invention binds to humanOX40 with a K_(D) of less than about 1 uM, about 900 nM, about 800 nM,about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM,about 200 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about60 nM, about 55 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM,about 30 nM, about 25 nM, about 20 nM, about 15 nM, about 10 nM, orabout 5 nM, or about 1 nM (as measured, for example, by surface plasmonresonance or biolayer interferometry). In embodiments, the chimericprotein binds to human OX40 with a K_(D) of less than about 1 nM, about900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80pM, about 70 pM, about 60 pM about 55 pM about 50 pM about 45 pM, about40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM,or about 10 pM, or about 1 pM (as measured, for example, by surfaceplasmon resonance or biolayer interferometry). In embodiments, thechimeric protein binds to human OX40 with a K_(D) of from about 200 pMto about 600 pM (as measured, for example, by surface plasmon resonanceor biolayer interferometry).

In embodiments, the chimeric protein may comprise an amino acid sequencehaving one or more amino acid mutations relative to any of the proteinsequences described herein. In embodiments, the chimeric proteincomprises a sequence that has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100or more amino acid mutations with respect to any one of the amino acidsequences of chimeric proteins disclosed herein.

In embodiments, the one or more amino acid mutations may beindependently selected from substitutions, insertions, deletions, andtruncations.

In embodiments, the amino acid mutations are amino acid substitutions,and may include conservative and/or non-conservative substitutions.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups: (1) hydrophobic: Met, Ala,Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3)acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influencechain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt a-helices.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above.

In embodiments, the substitutions may also include non-classical aminoacids (e.g. selenocysteine, pyrrolysine, N-formylmethionine β-alanine,GABA and δ-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers ofthe common amino acids, 2,4-diaminobutyric acid, α-amino isobutyricacid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx,6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme,citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as βmethyl amino acids, Cα-methyl amino acids, N α-methyl amino acids, and amino acid analogs ingeneral).

Mutations may also be made to the nucleotide sequences of the chimericproteins by reference to the genetic code, including taking into accountcodon degeneracy.

In embodiments, the present chimeric proteins may be variants describedherein, for instance, the present chimeric proteins may have a sequencehaving at least about 60%, or at least about 61%, or at least about 62%,or at least about 63%, or at least about 64%, or at least about 65%, orat least about 66%, or at least about 67%, or at least about 68%, or atleast about 69%, or at least about 70%, or at least about 71%, or atleast about 72%, or at least about 73%, or at least about 74%, or atleast about 75%, or at least about 76%, or at least about 77%, or atleast about 78%, or at least about 79%, or at least about 80%, or atleast about 81%, or at least about 82%, or at least about 83%, or atleast about 84%, or at least about 85%, or at least about 86%, or atleast about 87%, or at least about 88%, or at least about 89%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99%) sequence identity with the amino acid sequence of thepresent chimeric proteins, e.g. one or more of SEQ IDs Nos 5 and 6.

In embodiments, the chimeric protein comprises a linker. In embodiments,the linker comprising at least one cysteine residue capable of forming adisulfide bond. As described elsewhere herein, such at least onecysteine residue capable of forming a disulfide bond is, without wishingto be bound by theory, responsible for maintain a proper multimericstate of the chimeric protein and allowing for efficient production.

In embodiments, the chimeric protein of the present invention comprises:(a) a first domain comprising a portion of V-set and immunoglobulindomain-containing protein 8 (VSIG8), e.g., the extracellular domain ofVSIG8, that is capable of binding VISTA, (b) a second domain comprisinga portion of OX40L, e.g., the extracellular domain of OX40L, that iscapable of binding OX40, and optionally, (c) a linker linking the firstdomain and the second domain.

In embodiments, chimeric protein is a recombinant fusion protein, e.g.,a single polypeptide having the extracellular domains described herein(and, optionally a linker). For example, in embodiments, the chimericprotein is translated as a single unit in a cell. In embodiments,chimeric protein refers to a recombinant protein of multiplepolypeptides, e.g. multiple extracellular domains described herein, thatare linked to yield a single unit, e.g. in vitro (e.g. with one or moresynthetic linkers described herein). In embodiments, the chimericprotein is chemically synthesized as one polypeptide or each domain maybe chemically synthesized separately and then combined. In embodiments,a portion of the chimeric protein is translated and a portion ischemically synthesized.

In embodiments, the chimeric protein comprises a linker. In embodiments,the linker may be derived from naturally-occurring multi-domain proteinsor are empirical linkers as described, for example, in Chichili et al.,(2013), Protein Sci. 22(2):153-167, Chen et al., (2013), Adv Drug DelivRev. 65(10):1357-1369, the entire contents of which are herebyincorporated by reference. In embodiments, the linker may be designedusing linker designing databases and computer programs such as thosedescribed in Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369and Crasto et. al., (2000), Protein Eng. 13(5):309-312, the entirecontents of which are hereby incorporated by reference.

In embodiments, the linker is a synthetic linker such as PEG.

In embodiments, the linker comprises a polypeptide. In embodiments, thepolypeptide is less than about 500 amino acids long, about 450 aminoacids long, about 400 amino acids long, about 350 amino acids long,about 300 amino acids long, about 250 amino acids long, about 200 aminoacids long, about 150 amino acids long, or about 100 amino acids long.For example, the linker may be less than about 100, about 95, about 90,about 85, about 80, about 75, about 70, about 65, about 60, about 55,about 50, about 45, about 40, about 35, about 30, about 25, about 20,about 19, about 18, about 17, about 16, about 15, about 14, about 13,about 12, about 11, about 10, about 9, about 8, about 7, about 6, about5, about 4, about 3, or about 2 amino acids long. In embodiments, thelinker is flexible. In embodiments, the linker is rigid.

In embodiments, the linker is substantially comprised of glycine andserine residues (e.g. about 30%, or about 40%, or about 50%, or about60%, or about 70%, or about 80%, or about 90%, or about 95%, or about97%, or about 98%, or about 99%, or about 100% glycines and serines).

In embodiments, the linker comprises a hinge region of an antibody(e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g. IgG1,IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region, found inIgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer,allowing the Fab portion to move freely in space. In contrast to theconstant regions, the hinge domains are structurally diverse, varying inboth sequence and length among immunoglobulin classes and subclasses.For example, the length and flexibility of the hinge region varies amongthe IgG subclasses. The hinge region of IgG1 encompasses amino acids216-231 and, because it is freely flexible, the Fab fragments can rotateabout their axes of symmetry and move within a sphere centered at thefirst of two inter-heavy chain disulfide bridges. IgG2 has a shorterhinge than IgG1, with 12 amino acid residues and four disulfide bridges.The hinge region of IgG2 lacks a glycine residue, is relatively short,and contains a rigid poly-proline double helix, stabilized by extrainter-heavy chain disulfide bridges. These properties restrict theflexibility of the IgG2 molecule. IgG3 differs from the other subclassesby its unique extended hinge region (about four times as long as theIgG1 hinge), containing 62 amino acids (including 21 prolines and 11cysteines), forming an inflexible poly-proline double helix. In IgG3,the Fab fragments are relatively far away from the Fc fragment, givingthe molecule a greater flexibility. The elongated hinge in IgG3 is alsoresponsible for its higher molecular weight compared to the othersubclasses. The hinge region of IgG4 is shorter than that of IgG1 andits flexibility is intermediate between that of IgG1 and IgG2. Theflexibility of the hinge regions reportedly decreases in the orderIgG3>IgG1>IgG4>IgG2. In embodiments, the linker may be derived fromhuman IgG4 and contain one or more mutations to enhance dimerization(including S228P) or FcRn binding.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge region, the core region, and the lower hinge region. See Shin etal., 1992 Immunological Reviews 130:87. The upper hinge region includesamino acids from the carboxyl end of C_(H1) to the first residue in thehinge that restricts motion, generally the first cysteine residue thatforms an interchain disulfide bond between the two heavy chains. Thelength of the upper hinge region correlates with the segmentalflexibility of the antibody. The core hinge region contains theinter-heavy chain disulfide bridges, and the lower hinge region joinsthe amino terminal end of the C_(H2) domain and includes residues inC_(H2). Id. The core hinge region of wild-type human IgG1 contains thesequence CPPC (SEQ ID NO: 48) which, when dimerized by disulfide bondformation, results in a cyclic octapeptide believed to act as a pivot,thus conferring flexibility. In embodiments, the present linkercomprises, one, or two, or three of the upper hinge region, the coreregion, and the lower hinge region of any antibody (e.g., of IgG, IgA,IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2, IgG3, and IgG4,and IgA1 and IgA2)). The hinge region may also contain one or moreglycosylation sites, which include a number of structurally distincttypes of sites for carbohydrate attachment. For example, IgA1 containsfive glycosylation sites within a 17-amino-acid segment of the hingeregion, conferring resistance of the hinge region polypeptide tointestinal proteases, considered an advantageous property for asecretory immunoglobulin. In embodiments, the linker of the presentinvention comprises one or more glycosylation sites.

In embodiments, the linker comprises an Fc domain of an antibody (e.g.,of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2,IgG3, and IgG4, and IgA1 and IgA2)). In embodiments, the linkercomprises a hinge-CH2-CH3 Fc domain derived from a human IgG4 antibody.In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derivedfrom a human IgG1 antibody. In embodiments, the Fc domain exhibitsincreased affinity for and enhanced binding to the neonatal Fc receptor(FcRn). In embodiments, the Fc domain includes one or more mutationsthat increases the affinity and enhances binding to FcRn. Withoutwishing to be bound by theory, it is believed that increased affinityand enhanced binding to FcRn increases the in vivo half-life of thepresent chimeric proteins.

In embodiments, the Fc domain linker contains one or more amino acidsubstitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311,416, 428, 433 or 434 (in accordance with Kabat numbering, as in as inKabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National

Institutes of Health, Bethesda, Md. (1991) expressly incorporatedherfein by reference), or equivalents thereof. In embodiments, the aminoacid substitution at amino acid residue 250 is a substitution withglutamine. In embodiments, the amino acid substitution at amino acidresidue 252 is a substitution with tyrosine, phenylalanine, tryptophanor threonine. In embodiments, the amino acid substitution at amino acidresidue 254 is a substitution with threonine. In embodiments, the aminoacid substitution at amino acid residue 256 is a substitution withserine, arginine, glutamine, glutamic acid, aspartic acid, or threonine.In embodiments, the amino acid substitution at amino acid residue 308 isa substitution with threonine. In embodiments, the amino acidsubstitution at amino acid residue 309 is a substitution with proline.In embodiments, the amino acid substitution at amino acid residue 311 isa substitution with serine. In embodiments, the amino acid substitutionat amino acid residue 385 is a substitution with arginine, asparticacid, serine, threonine, histidine, lysine, alanine or glycine. Inembodiments, the amino acid substitution at amino acid residue 386 is asubstitution with threonine, proline, aspartic acid, serine, lysine,arginine, isoleucine, or methionine. In embodiments, the amino acidsubstitution at amino acid residue 387 is a substitution with arginine,proline, histidine, serine, threonine, or alanine. In embodiments, theamino acid substitution at amino acid residue 389 is a substitution withproline, serine or asparagine. In embodiments, the amino acidsubstitution at amino acid residue 416 is a substitution with serine. Inembodiments, the amino acid substitution at amino acid residue 428 is asubstitution with leucine. In embodiments, the amino acid substitutionat amino acid residue 433 is a substitution with arginine, serine,isoleucine, proline, or glutamine. In embodiments, the amino acidsubstitution at amino acid residue 434 is a substitution with histidine,phenylalanine, or tyrosine.

In embodiments, the Fc domain linker (e.g., comprising an IgG constantregion) comprises one or more mutations such as substitutions at aminoacid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabatnumbering, as in as in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) expressly incorporated hereinby reference). In embodiments, the IgG constant region includes a tripleM252Y/S254T/T256E mutation or YTE mutation. In embodiments, the IgGconstant region includes a triple H433K/N434F/Y436H mutation or KFHmutation. In embodiments, the IgG constant region includes an YTE andKFH mutation in combination.

In embodiments, the modified humanized antibodies of the inventioncomprise an IgG constant region that contains one or more mutations atamino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435 (inaccordance with Kabat numbering, as in as in Kabat, et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) expresslyincorporated herein by reference). Illustrative mutations include T250Q,M428L, T307A, E380A, 1253A, H310A, M428L, H433K, N434A, N434F, N434S,and H435A. In embodiments, the IgG constant region comprises aM428L/N434S mutation or LS mutation. In embodiments, the IgG constantregion comprises a M428L/N434S mutation or LS mutation. In embodiments,the IgG constant region comprises a T250Q/M428L mutation or QL mutation.In embodiments, the IgG constant region comprises an N434A mutation. Inembodiments, the IgG constant region comprises a T307A/E380A/N434Amutation or AAA mutation. In embodiments, the IgG constant regioncomprises an 1253A/H310A/H435A mutation or IHH mutation. In embodiments,the IgG constant region comprises a H433K/N434F mutation. Inembodiments, the IgG constant region comprises a M252Y/S254T/T256E and aH433K/N434F mutation in combination.

Additional exemplary mutations in the IgG constant region are described,for example, in Robbie, et al., Antimicrobial Agents and Chemotherapy(2013), 57(12):6147-6153, Dall'Acqua et al., JBC (2006),281(33):23514-24, Dall'Acqua et al., Journal of Immunology (2002),169:5171-80, Ko et al. Nature (2014) 514:642-645, Grevys et al. Journalof Immunology. (2015), 194(11):5497-508, and U.S. Pat. No. 7,083,784,the entire contents of which are hereby incorporated by reference.

In embodiments, the Fc domain in a linker comprises the amino acidsequence of SEQ ID NO: 25 (see the below table), or at least 90%, or93%, or 95%, or 97%, or 98%, or 99% identity thereto. In embodiments,mutations are made to SEQ ID NO: 25 to increase stability and/orhalf-life. For instance, in embodiments, the Fc domain in a linkercomprises the amino acid sequence of SEQ ID NO: 26 (see the belowtable), or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identitythereto. An illustrative Fc stabilizing mutant is S228P. Illustrative Fchalf-life extending mutants are T250Q, M428L, V308T, L309P, and Q311Sand the present linkers may comprise 1, or 2, or 3, or 4, or 5 of thesemutants. In embodiments, the Fc domain in a linker comprises the aminoacid sequence of SEQ ID NO: 27 (see the below table), or at least 90%,or 93%, or 95%, or 97%, or 98%, or 99% identity thereto.

Further, one or more joining linkers may be employed to connect an Fcdomain in a linker (e.g. one of SEQ ID NOs: 25, 26, or 27 or at least90%, or 93%, or 95%, or 97%, or 98%, or 99% identity thereto) and theextracellular domains. For example, any one of SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, orvariants thereof may connect an extracellular domain as described hereinand a linker as described herein. Optionally, any one of SEQ ID NOs:28-74, or variants thereof are displaced between an extracellular domainas described herein and a linker as described herein.

The amino acid sequence of SEQ ID NOs: 25-74 are provided in the Table 1below.

TABLE 1 Illustrative linkers (Fc domain linkers and joining linkers)SEQ ID NO. Sequence 25APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 26APEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTTPHSDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVLHEALHNHYTQKSLSLSLGK 27APEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGK 28 SKYGPPCPSCP 29 SKYGPPCPPCP 30SKYGPP 31 IEGRMD 32 GGGVPRDCG 33 IEGRMDGGGGAGGGG 34 GGGSGGGS 35GGGSGGGGSGGG 36 EGKSSGSGSESKST 37 GGSG 38 GGSGGGSGGGSG 39EAAAKEAAAKEAAAK 40 EAAAREAAAREAAAREAAAR 41 GGGGSGGGGSGGGGSAS 42GGGGAGGGG 43 GS or GGS or LE 44 GSGSGS 45 GSGSGSGSGS 46 GGGGSAS 47APAPAPAPAPAPAPAPAPAP 48 CPPC 49 GGGGS 50 GGGGSGGGGS 51 GGGGSGGGGSGGGGS52 GGGGSGGGGSGGGGSGGGGS 53 GGGGSGGGGSGGGGSGGGGSGGGGS 54GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 55 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 56GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 57 GGSGGSGGGGSGGGGS 58 GGGGGGGG59 GGGGGG 60 EAAAK 61 EAAAKEAAAK 62 EAAAKEAAAKEAAAK 63 AEAAAKEAAAKA 64AEAAAKEAAAKEAAAKA 65 AEAAAKEAAAKEAAAKEAAAKA 66AEAAAKEAAAKEAAAKEAAAKEAAAKA 67AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA 68 PAPAP 69KESGSVSSEQLAQFRSLD 70 GSAGSAAGSGEF 71 GGGSE 72 GSESG 73 GSEGS 74GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS

In embodiments, the joining linker substantially comprises glycine andserine residues (e.g., about 30%, or about 40%, or about 50%, or about60%, or about 70%, or about 80%, or about 90%, or about 95%, or about97%, or about 98%, or about 99%, or about 100% glycines and serines).For example, in embodiments, the joining linker is (Gly₄Ser)_(n), wheren is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ IDNO: 49 to SEQ ID NO: 56, respectively). In embodiments, the joininglinker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 57). Additionalillustrative joining linkers include, but are not limited to, linkershaving the sequence LE, (Gly)₈ (SEQ ID NO: 58), (Gly)₆ (SEQ ID NO: 59),(EAAAK)_(n) (n=1-3) (SEQ ID NO: 60-SEQ ID NO: 62), A(EAAAK)_(n)A (n=2-5)(SEQ ID NO: 63-SEQ ID NO: 66), A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO: 67),PAPAP (SEQ ID NO: 68), KESGSVSSEQLAQFRSLD (SEQ ID NO: 69), GSAGSAAGSGEF(SEQ ID NO: 70), and (XP)_(n), with X designating any amino acid, e.g.,Ala, Lys, or Glu. In embodiments, the joining linker is GGS.

In embodiments, the joining linker is one or more of GGGSE (SEQ ID NO:71), GSESG (SEQ ID NO: 72), GSEGS (SEQ ID NO: 73),GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 74), and a joininglinker of randomly placed G, S, and E every 4 amino acid intervals.

In embodiments, a chimeric protein comprises a modular linker as shownin FIG. 22.

In embodiments, the linker may be functional. For example, withoutlimitation, the linker may function to improve the folding and/orstability, improve the expression, improve the pharmacokinetics, and/orimprove the bioactivity of the present chimeric protein. In anotherexample, the linker may function to target the chimeric protein to aparticular cell type or location.

In embodiments, the chimeric protein exhibits enhanced stability andprotein half-life. In embodiments, the chimeric protein binds to FcRnwith high affinity. In embodiments, the chimeric protein may bind toFcRn with a K_(D) of about 1 nM to about 80 nM. For example, thechimeric protein may bind to FcRn with a K_(D) of about 1 nM, about 2nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM,about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about55 nM, about 60 nM, about 65 nM, about 70 nM, about 71 nM, about 72 nM,about 73 nM, about 74 nM, about 75 nM, about 76 nM, about 77 nM, about78 nM, about 79 nM, or about 80 nM. In embodiments, the chimeric proteinmay bind to FcRn with a K_(D) of about 9 nM. In embodiments, thechimeric protein does not substantially bind to other Fc receptors (i.e.other than FcRn) with effector function.

In embodiments, a chimeric protein having the formula ECD 1—JoiningLinker 1—Fc Domain—Joining Linker 2—ECD 2, in which ECD 1 is VSIG8 andECD 2 is OX40L may be referred to in the present disclosure asVSIG8-Fc-OX40L. In embodiments, the chimeric protein lacks one or bothjoining linkers; such a chimeric protein may also be referred to in thepresent disclosure as VSIG8-Fc-OX40L. These chimeric proteins may lackone or both of the joining linkers. Exemplary Joining Linker 1s, FcDomains, and Joining Linker 2s are described above in Table 1; modularlinkers useful for forming chimeric proteins and comprising specificJoining Linker 1s, Fc Domains, and Joining Linker 2s are shown in FIG.22.

In embodiments, the method generates a memory response which may, e.g.be capable of preventing relapse.

In embodiments, a chimeric protein is a fusion protein having theformula N terminus—(a)—(b)—(c)—C terminus, in which (a) is VSIG8, (b) isa linker comprising at least a portion of a Fc domain, and (c) is OX40Lmay be referred to in the present disclosure as VSIG8-Fc-OX40L.

In embodiments, a chimeric protein is optimized for/directed to murineligands/receptors; an example of such a chimeric protein is murineVSIG8-Fc-OX40L, which is also referred herein as mVSIG8-Fc-OX40L.

In embodiments, a chimeric protein is optimized for/directed to humanligands/receptors; an example of such a chimeric protein is humanVSIG8-Fc-OX40L, which is also referred herein as hVSIG8-Fc-OX40L.

In embodiments, the present chimeric protein targets the VISTANSIG8immune inhibitory signaling pathway. In embodiments, the chimericprotein disrupts, blocks, reduces, and/or inhibits the transmission ofan immune inhibitory signal mediated by binding of VISTA to VSIG8. Inembodiments, an immune inhibitory signal refers to a signal thatdiminishes or eliminates an immune response. For example, in the contextof oncology, such signals may diminish or eliminate antitumor immunity.Under normal physiological conditions, inhibitory signals are useful inthe maintenance of self-tolerance (e.g. prevention of autoimmunity) andalso to protect tissues from damage when the immune system is respondingto pathogenic infection. For instance, without limitation, an immuneinhibitory signal may be identified by detecting an increase in cellularproliferation, cytokine production, cell killing activity or phagocyticactivity when such an inhibitory signal is blocked.

In embodiments, the present chimeric protein disrupts, blocks, reduces,and/or inhibits the transmission of an immune inhibitory signal mediatedby the binding of VISTA, or other binding partners, to VSIG8. Inembodiments, the chimeric protein binds to VISTA but disrupts, blocks,reduces, and/or inhibits the inhibitory signal transmission to an immunecell (e.g. a T cell). In embodiments, the chimeric protein acts on, forexample, a lymphocyte cell that expresses VISTA and disrupts, blocks,reduces, and/or inhibits inhibitory signal transmission to an immunecell (e.g. a T cell).

In embodiments, the present chimeric proteins are capable of, or finduse in methods comprising, reducing, disrupting, or eliminating thebinding of the immune inhibitory receptor/ligand pair, VISTA/VSIG8. Inembodiments, the present chimeric protein blocks, reduces, and/orinhibits VSIG8 and/or the binding of VSIG8 with VISTA or with otherbinding partners.

In embodiments, the present chimeric protein targets an immunestimulatory signal mediated by the binding of OX40L to OX40. Inembodiments, the chimeric protein enhances, increases, and/or stimulatesthe transmission of an immune stimulatory signal mediated by binding ofOX40L to OX40. In embodiments, an immune stimulatory signal refers to asignal that enhances an immune response. For example, in the context ofoncology, such signals may enhance antitumor immunity. For instance,without limitation, immune stimulatory signal may be identified bydirectly stimulating proliferation, cytokine production, killingactivity or phagocytic activity of leukocytes, including subsets of Tcells.

In embodiments, the present chimeric protein enhances, increases, and/orstimulates the transmission of an immune stimulatory signal mediated bythe binding of OX40L to OX40. In embodiments, the present chimericprotein comprising the extracellular domain of OX40L acts on an immunecell (e.g., a T cell) that expresses OX40 and enhances, increases,and/or stimulates stimulatory signal transmission to the immune cell(e.g., a T cell).

In embodiments, the present chimeric proteins are capable of, or finduse in methods comprising, stimulating or enhancing the binding of theimmune stimulatory receptor/ligand pair, OX-40:OX40L. In embodiments,the present chimeric protein increases and/or stimulates OX40 and/or thebinding of OX40 with one or more of OX40L.

In embodiments, a chimeric protein comprises an extracellular domain oftype II protein, other than OX40L. Exemplary type II proteins include4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A, and TRAIL. The presentinvention further includes chimeric proteins and methods using thefollowing chimeric proteins: VISIG8/4-1BBL, VISIG8/CD30L, VISIG8/CD40L,VISIG8/FasL, VISIG8/GITRL, VISIG8/LIGHT, VISIG8/TL1A, and VISIG8/TRAIL.In embodiments, the chimeric protein has a general structure of one ofVISIG8-Fc-4-1BBL, VISIG8-Fc-CD30L, VISIG8-Fc-CD40L, VISIG8-Fc-FasL,VISIG8-Fc-GITRL, VISIG8-Fc-LIGHT, VISIG8-Fc-TL1A, and VISIG8-Fc-TRAIL.

The amino acid sequence for 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT,TL1A, and TRAIL, respectively, comprises SEQ ID NO: 7, 9, 11, 13, 15,17, 21, and 23.

In embodiments, a chimeric protein comprises the extracellular domain ofone of 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A, and TRAIL which,respectively, comprises SEQ ID NO: 8, 10, 12, 14, 16, 18, 22, and 24. Inembodiments, the present chimeric proteins may comprise theextracellular domain of 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A,or TRAIL as described herein, or a variant or a functional fragmentthereof. For instance, the chimeric protein may comprise a sequence ofthe extracellular domain of 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT,TL1A, or TRAIL as provided above, or a variant or functional fragmentthereof having at least about 60%, or at least about 61%, or at leastabout 62%, or at least about 63%, or at least about 64%, or at leastabout 65%, or at least about 66%, or at least about 67%, or at leastabout 68%, or at least about 69%, or at least about 70%, or at leastabout 71%, or at least about 72%, or at least about 73%, or at leastabout 74%, or at least about 75%, or at least about 76%, or at leastabout 77%, or at least about 78%, or at least about 79%, or at leastabout 80%, or at least about 81%, or at least about 82%, or at leastabout 83%, or at least about 84%, or at least about 85%, or at leastabout 86%, or at least about 87%, or at least about 88%, or at leastabout 89%, or at least about 90%, or at least about 91%, or at leastabout 92%, or at least about 93%, or at least about 94%, or at leastabout 95%, or at least about 96%, or at least about 97%, or at leastabout 98%, or at least about 99% sequence identity with the amino acidsequence of the extracellular domain of 4-1BBL, CD30L, CD40L, FasL,GITRL, LIGHT, TL1A, or TRAIL as described herein.

In embodiments, the present chimeric proteins may comprises variants ofthe extracellular domains described herein, for instance, a sequencehaving at least about 60%, or at least about 61%, or at least about 62%,or at least about 63%, or at least about 64%, or at least about 65%, orat least about 66%, or at least about 67%, or at least about 68%, or atleast about 69%, or at least about 70%, or at least about 71%, or atleast about 72%, or at least about 73%, or at least about 74%, or atleast about 75%, or at least about 76%, or at least about 77%, or atleast about 78%, or at least about 79%, or at least about 80%, or atleast about 81%, or at least about 82%, or at least about 83%, or atleast about 84%, or at least about 85%, or at least about 86%, or atleast about 87%, or at least about 88%, or at least about 89%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99%) sequence identity with the known amino acid or nucleicacid sequence of the extracellular domains, e.g. human extracellulardomains, e.g. one or more of SEQ IDs NOs: 2, 4, 8, 10, 12, 14, 16, 18,20, 22, and 24.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of VSIG8 (SEQ ID NO: 2).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of OX40L (SEQ ID NO: 4).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of CD40L (SEQ ID NO: 12).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of VSIG8 (SEQ ID NO: 2) and the extracellulardomain of OX40L (SEQ ID NO: 4).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of VSIG8 (SEQ ID NO: 2) and the extracellulardomain of CD40L (SEQ ID NO: 12).

In embodiments, the chimeric protein of the present invention comprisesthe hinge-CH2-CH3 domain from a human IgG4 antibody sequence (SEQ ID NO:25, 26, or 27.

In embodiments, a chimeric protein comprises a modular linker as shownin FIG. 22.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of VSIG8 and the extracellular domain of OX40L,using the hinge-CH2-CH3 domain from a human IgG4 antibody sequence as alinker (this VSIG8-Fc-OX40L chimera is SEQ ID NO: 5).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of VSIG8 and the extracellular domain of CD40L,using the hinge-CH2-CH3 domain from a human IgG4 antibody sequence as alinker (this VSIG8-Fc-CD40L chimera is SEQ ID NO: 6).

In embodiments, the chimeric protein of the invention delivers an immunestimulation to an immune cell (e.g., a T cell) while masking immuneinhibitory signals. In embodiments, the chimeric protein deliverssignals that have the net result of immune activation (e.g., T cellactivation).

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, promoting immune activation (e.g. againsttumors). In embodiments, the present chimeric proteins are capable of,and can be used in methods comprising, suppressing immune inhibition(e.g. that allows tumors to survive). In embodiments, the presentchimeric proteins provide improved immune activation and/or improvedsuppression of immune inhibition due to the proximity of signaling thatis provided by the chimeric nature of the constructs.

In embodiments, the present chimeric proteins are capable of, or can beused in methods comprising, modulating the amplitude of an immuneresponse, e.g. modulating the level of effector output. In embodiments,e.g. when used for the treatment of a cancer and/or an inflammatorydisease, the present chimeric proteins alter the extent of immunestimulation as compared to immune inhibition to increase the amplitudeof a T cell response, including, without limitation, stimulatingincreased levels of cytokine production, proliferation or target killingpotential.

In embodiments, the present chimeric proteins are capable of, or finduse in methods involving, masking an inhibitory ligand and replacingthat immune inhibitory ligand with an immune stimulatory ligand. Forexample, the present chimeric protein construct comprising (i) theextracellular domain of VSIG8 and (ii) extracellular domain of OX40L,allows for the disruption of an inhibitory VISTA/VSIG8 signal andreplacing it with a stimulating OX40L/OX40 signal. Accordingly, thepresent chimeric proteins, in embodiments are capable of, or find use inmethods involving, reducing or eliminating an inhibitory immune signaland/or increasing or activating an immune stimulatory signal. Forexample, a lymphocyte or other cell bearing an inhibitory signal (andthus evading an immune response) may be substituted for a positivesignal binding on a T cell that can then attack a tumor cell.Accordingly, in embodiments, an inhibitory immune signal is masked bythe present constructs and a stimulatory immune signal is activated.Such beneficial properties are enhanced by the single construct approachof the present chimeric proteins. For instance, the signal replacementcan be effected nearly simultaneously and the signal replacement istailored to be local at a site of clinical importance (e.g. the tumormicroenvironment). In embodiments, the construct localizes positiveimmune-stimulatory signals near blockade or inhibitory signals, tobetter couple these functionalities. This local coupling effect mayexplain the superior performance of the VSIG8-Fc-OX40L constructs ascompared to combinations of VISTA blocking and OX40 agonist antibodies,for example.

In embodiments, the present chimeric proteins are capable of, or finduse in methods involving, enhancing, restoring, promoting and/orstimulating immune modulation. In embodiments, the present chimericproteins described herein, restore, promote and/or stimulate theactivity or activation of one or more immune cells against tumor cellsincluding, but not limited to: T cells, cytotoxic T lymphocytes, Thelper cells, natural killer (NK) cells, natural killer T (NKT) cells,anti-tumor macrophages (e.g. M1 macrophages), B cells, and dendriticcells. In embodiments, the present chimeric proteins enhance, restore,promote and/or stimulate the activity and/or activation of T cells,including, by way of a non-limiting example, activating and/orstimulating one or more T-cell intrinsic signals, including apro-survival signal; an autocrine or paracrine growth signal; a p38MAPK-, ERK-, STAT-, JAK-, AKT- or PI3K-mediated signal; ananti-apoptotic signal; and/or a signal promoting and/or necessary forone or more of: proinflammatory cytokine production or T cell migrationor T cell tumor infiltration.

In embodiments, the present chimeric proteins are capable of, or finduse in methods involving, causing an increase of one or more of T cells(including without limitation cytotoxic T lymphocytes, T helper cells,natural killer T (NKT) cells), B cells, natural killer (NK) cells,natural killer T (NKT) cells, dendritic cells, monocytes, andmacrophages (e.g. one or more of M1 and M2) into a tumor or the tumormicroenvironment. In embodiments, the chimeric protein enhances therecognition of tumor antigens by CD8+ T cells, particularly those Tcells that have infiltrated into the tumor microenvironment.

In embodiments, the present chimeric proteins are capable of, or finduse in methods involving, inhibiting and/or causing a decrease inrecruitment of immunosuppressive cells (e.g. myeloid-derived suppressorcells (MDSCs), regulatory T cells (Tregs), tumor associated neutrophils(TANs), M2 macrophages, and tumor associated macrophages (TAMs), andparticularly within the tumor and/or tumor microenvironment (TME). Inembodiments, the present therapies may alter the ratio of M1 versus M2macrophages in the tumor site and/or TME to favor M1 macrophages.

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, inhibiting and/or reducing T cellinactivation and/or immune tolerance to a tumor, comprisingadministering an effective amount of a chimeric protein described hereinto a subject. In embodiments, the present chimeric proteins are able toincrease the serum levels of various cytokines including, but notlimited to, one or more of IFNγ, INFα, IL-2, IL-4, IL-5, IL-6, IL-9,IL-10, IL-13, IL-17A, IL-17F, and IL-22. In embodiments, the presentchimeric proteins are capable of enhancing IL-2, IL-4, IL-5, IL-10,IL-13, IL-17A, IL-22, INFα or IFNγ in the serum of a treated subject.Detection of such a cytokine response may provide a method to determinethe optimal dosing regimen for the indicated chimeric protein.

In embodiments, the present chimeric proteins inhibit, block and/orreduce cell death of an anti-tumor CD8+ and/or CD4+ T cell; orstimulate, induce, and/or increase cell death of a pro-tumor T cell. Tcell exhaustion is a state of T cell dysfunction characterized byprogressive loss of proliferative and effector functions, culminating inclonal deletion. Accordingly, a pro-tumor T cell refers to a state of Tcell dysfunction that arises during many chronic infections,inflammatory diseases, and cancer. This dysfunction is defined by poorproliferative and/or effector functions, sustained expression ofinhibitory receptors and a transcriptional state distinct from that offunctional effector or memory T cells. Exhaustion prevents optimalcontrol of infection and tumors. In addition, an anti-tumor CD8+ and/orCD4+ T cell refers to T cells that can mount an immune response to atumor. Illustrative pro-tumor T cells include, but are not limited to,Tregs, CD430 and/or CD8+ T cells expressing one or more checkpointinhibitory receptors, Th2 cells and Th17 cells. Checkpoint inhibitoryreceptors refer to receptors expressed on immune cells that prevent orinhibit uncontrolled immune responses.

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, increasing a ratio of effector T cells toregulatory T cells. Illustrative effector T cells include ICOS⁺ effectorT cells; cytotoxic T cells (e.g. αβ TCR, CD3⁺, CD8⁺, CD45RO⁺); CD4⁺effector T cells (e.g. αβ TCR, CD3⁺, CD4⁺, CCR7⁺, CD62Lhi,IL-7R/CD127⁺); CD8⁺ effector T cells (e.g. αβTCR, CD3⁺, CD8⁺, CCR7⁺,CD62Lhi, IL-7R/CD127⁺); effector memory T cells (e.g. CD62Llow, CD44⁺,TCR, CD3⁺, IL-7R/CD127⁺, IL-15R⁺, CCR7low); central memory T cells (e.g.CCR7⁺, CD62L⁺, CD27⁺; or CCR7hi, CD44⁺, CD62Lhi, TCR, CD3⁺,IL-7R/CD127⁺, IL-15R⁺); CD62L⁺ effector T cells; CD8⁺ effector memory Tcells (TEM) including early effector memory T cells (CD27⁺CD62L⁻) andlate effector memory T cells (CD27⁻ CD62L⁻) (TemE and TemL,respectively); CD127(⁺)CD25(low/−) effector T cells; CD127(⁻)CD25(⁻)effector T cells; CD8⁺ stem cell memory effector cells (TSCM) (e.g.CD44(low)CD62L(high)CD122(high)sca(⁺)); TH1 effector T-cells (e.g.CXCR3⁺, CXCR6⁺ and CCR5⁺; or αβ TCR, CD3⁺, CD4⁺, IL-12R⁺, IFNγR⁺,CXCR3⁺), TH2 effector T cells (e.g. CCR3⁺, CCR4⁺ and CCR8⁺; or αβ TCR,CD3⁺, CD4⁺, IL-4R⁺, IL-33R⁺, CCR4⁺, IL-17RB⁺, CRTH2⁺); TH9 effector Tcells (e.g. αβ TCR, CD3⁺, CD4⁺); TH17 effector T cells (e.g. αβ TCR,CD3⁺, CD4⁺, IL-23R⁺, CCR6⁺, IL-1R⁺); CD4⁺CD45RO⁺CCR7⁺ effector T cells,CD4⁺CD45RO⁺CCR7(⁻) effector T cells; and effector T cells secretingIL-2, IL-4 and/or IFN-γ. Illustrative regulatory T cells include ICOS⁺regulatory T cells, CD4⁺CD25⁺FOXP3⁺ regulatory T cells, CD4⁺CD25⁺regulatory T cells, CD4⁺CD25⁻ regulatory T cells, CD4⁺CD25 highregulatory T cells, TIM-3⁺PD-1⁺ regulatory T cells, lymphocyteactivation gene-3 (LAG-3)⁺ regulatory T cells, CTLA-4/CD152⁺ regulatoryT cells, neuropilin-1 (Nrp-1)⁺ regulatory T cells, CCR4⁺CCR8⁺ regulatoryT cells, CD62L (L-selectin)⁺ regulatory T cells, CD45RBlow regulatory Tcells, CD127low regulatory T cells, LRRC32/GARP⁺ regulatory T cells,CD39⁺ regulatory T cells, GITR⁺ regulatory T cells, LAP⁺ regulatory Tcells, 1B11⁺ regulatory T cells, BTLA⁺ regulatory T cells, type 1regulatory T cells (Tr1 cells), T helper type 3 (Th3) cells, regulatorycell of natural killer T cell phenotype (NKTregs), CD8⁺ regulatory Tcells, CD8⁺CD28⁻ regulatory T cells and/or regulatory T-cells secretingIL-10, IL-35, TGF-62 , TNF-α, Galectin-1, IFN-γ and/or MCP1.

In embodiments, the chimeric protein of the invention causes an increasein effector T cells (e.g., CD4+CD25− T cells). In embodiments, thechimeric protein causes a decrease in regulatory T cells (e.g.,CD4+CD25− T cells).

In embodiments, the chimeric protein generates a memory response whichmay, e.g., be capable of preventing relapse or protecting the animalfrom a recurrence and/or preventing, or reducing the likelihood of,metastasis. Thus, an animal treated with the chimeric protein is laterable to attack tumor cells and/or prevent development of tumors whenexposed to the relevant antigen after an initial treatment with thechimeric protein. Accordingly, a chimeric protein of the presentinvention stimulates both active tumor destruction and also immunerecognition of tumor antigens, which are essential in programming amemory response capable of preventing relapse.

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, transiently stimulating effector immunecells for no longer than about 12 hours, about 24 hours, about 48 hours,about 72 hours or about 96 hours or about 1 week or about 2 weeks. Inembodiments, the present chimeric proteins are capable of, and can beused in methods comprising, transiently depleting or inhibitingregulatory or immune suppressive cells for no longer than about 12hours, about 24 hours, about 48 hours, about 72 hours or about 96 hoursor about 1 week or about 2 weeks. In embodiments, the transientstimulation of effector T cells and/or transient depletion or inhibitionof immune inhibitory cells occurs substantially in a patient'sbloodstream or in a particular tissue/location including lymphoidtissues such as for example, the bone marrow, lymph-node, spleen,thymus, mucosa-associated lymphoid tissue (MALT), non-lymphoid tissues,or in the tumor microenvironment.

In embodiments, the present chimeric proteins provide advantagesincluding, without limitation, ease of use and ease of production. Thisis because two distinct immunotherapy agents are combined into a singleproduct which allows for a single manufacturing process instead of twoindependent manufacturing processes. In addition, administration of asingle agent instead of two separate agents allows for easieradministration and greater patient compliance.

In embodiments, the present chimeric protein is producible in amammalian host cell as a secretable and fully functional singlepolypeptide chain.

In embodiments, the present chimeric protein unexpectedly providesbinding of the extracellular domain components to their respectivebinding partners with slow off rates (Kd or K_(off)). In embodiments,this provides an unexpectedly long interaction of the receptor to ligandand vice versa. Such an effect allows for a sustained negative signalmasking effect. Further, in embodiments, this delivers a longer positivesignal effect, e.g. to allow an effector cell to be adequatelystimulated for an anti-tumor effect. For example, the present chimericprotein, e.g. via the long off rate binding allows sufficient signaltransmission to provide T cell proliferation and allow for anti-tumorattack. By way of further example, the present chimeric protein, e.g.via the long off rate binding allows sufficient signal transmission toprovide release of stimulatory signals, such as, for example, cytokines.

The stable synapse of cells promoted by the present agents (e.g. cellsbearing negative signals and a T cell that can be stimulated to attackthe tumor) provides spatial orientation to favor tumor reduction—such aspositioning the T cells to attack tumor cells and/or stericallypreventing the tumor cell from delivering negative signals, includingnegative signals beyond those masked by the chimeric protein of theinvention.

In embodiments, this provides longer on-target (e.g. intra-tumoral)half-life (t_(1/2)) as compared to serum t_(1/2) of the chimericproteins. Such properties could have the combined advantage of reducingoff-target toxicities associated with systemic distribution of thechimeric proteins.

In embodiments, the present chimeric proteins provide a biphasic immuneeffect that provides rapid and latent response. For example, direct Tcell costimulation through OX40 or inhibition of signaling through VSIG8may lead to an immediate enhancement of anti-tumor activity mediated byT cells (or other immune cells) and enhance short-term control of tumorgrowth and rejection. In embodiments, the initial dual interactionbetween immune cells and VSIG8-Fc-OX40L may alter the strength orquality of immune priming, and contribute to enhanced generation oflong-lived memory T cell immunity. Such an event could be made evidentin tumor models by enhanced long-term control, or equilibrium, ofestablished tumors. In some cases, an immune response programmed bytreatment with VSIG8-Fc-OX40L may contribute to delayed tumorrejections, at times long after administration of the molecule.

Further, in embodiments, the present chimeric proteins providesynergistic therapeutic effects as it allows for improved site-specificinterplay of two immunotherapy agents.

In embodiments, the present chimeric proteins provide the potential forreducing off-site and/or systemic toxicity.

In embodiments, the present chimeric proteins provide reducedside-effects, e.g., GI complications, relative to currentimmunotherapies, e.g., antibodies directed to checkpoint moleclues asdescribed herein. Illustrative GI complications include abdominal pain,appetite loss, autoimmune effects, constipation, cramping, dehydration,diarrhea, eating problems, fatigue, flatulence, fluid in the abdomen orascites, gastrointestinal (GI) dysbiosis, GI mucositis, inflammatorybowel disease, irritable bowel syndrome (IBS-D and IBS-C), nausea, pain,stool or urine changes, ulcerative colitis, vomiting, weight gain fromretaining fluid, and/or weakness.

Diseases; Methods of Treatment, and Patient Selections

In embodiments, the present invention pertains to cancers and/or tumors;for example, the treatment or prevention of cancers and/or tumors. Asdescribed elsewhere herein, the treatment of cancer may involve Inembodiments, modulating the immune system with the present chimericproteins to favor immune stimulation over immune inhibition.

Cancers or tumors refer to an uncontrolled growth of cells and/orabnormal increased cell survival and/or inhibition of apoptosis whichinterferes with the normal functioning of the bodily organs and systems.

Included are benign and malignant cancers, polyps, hyperplasia, as wellas dormant tumors or micrometastases. Also, included are cells havingabnormal proliferation that is not impeded by the immune system (e.g.virus infected cells). The cancer may be a primary cancer or ametastatic cancer. The primary cancer may be an area of cancer cells atan originating site that becomes clinically detectable, and may be aprimary tumor. In contrast, the metastatic cancer may be the spread of adisease from one organ or part to another non-adjacent organ or part.The metastatic cancer may be caused by a cancer cell that acquires theability to penetrate and infiltrate surrounding normal tissues in alocal area, forming a new tumor, which may be a local metastasis. Thecancer may also be caused by a cancer cell that acquires the ability topenetrate the walls of lymphatic and/or blood vessels, after which thecancer cell is able to circulate through the bloodstream (thereby beinga circulating tumor cell) to other sites and tissues in the body. Thecancer may be due to a process such as lymphatic or hematogeneousspread. The cancer may also be caused by a tumor cell that comes to restat another site, re-penetrates through the vessel or walls, continues tomultiply, and eventually forms another clinically detectable tumor. Thecancer may be this new tumor, which may be a metastatic (or secondary)tumor.

The cancer may be caused by tumor cells that have metastasized, whichmay be a secondary or metastatic tumor. The cells of the tumor may belike those in the original tumor. As an example, if a breast cancer orcolon cancer metastasizes to the liver, the secondary tumor, whilepresent in the liver, is made up of abnormal breast or colon cells, notof abnormal liver cells. The tumor in the liver may thus be a metastaticbreast cancer or a metastatic colon cancer, not liver cancer.

The cancer may have an origin from any tissue. The cancer may originatefrom melanoma, colon, breast, or prostate, and thus may be made up ofcells that were originally skin, colon, breast, or prostate,respectively. The cancer may also be a hematological malignancy, whichmay be leukemia or lymphoma. The cancer may invade a tissue such asliver, lung, bladder, or intestinal.

Representative cancers and/or tumors of the present invention include,but are not limited to, a basal cell carcinoma, biliary tract cancer;bladder cancer; bone cancer; brain and central nervous system cancer;breast cancer; cancer of the peritoneum; cervical cancer;choriocarcinoma; colon and rectum cancer; connective tissue cancer;cancer of the digestive system; endometrial cancer; esophageal cancer;eye cancer; cancer of the head and neck; gastric cancer (includinggastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma;intra-epithelial neoplasm; kidney or renal cancer; larynx cancer;leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavitycancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreaticcancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectalcancer; cancer of the respiratory system; salivary gland carcinoma;sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicularcancer; thyroid cancer; uterine or endometrial cancer; cancer of theurinary system; vulval cancer; lymphoma including Hodgkin's andnon-Hodgkin's lymphoma, as well as B-cell lymphoma (including lowgrade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL)NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL;high grade immunoblastic NHL; high grade lymphoblastic NHL; high gradesmall non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (such as that associated with brain tumors),and Meigs' syndrome.

In embodiments, the chimeric protein is used to treat a subject that hasa treatment-refractory cancer. In embodiments, the chimeric protein isused to treat a subject that is refractory to one or moreimmune-modulating agents. For example, in embodiments, the chimericprotein is used to treat a subject that presents no response totreatment, or even progress, after 12 weeks or so of treatment. Forinstance, in embodiments, the subject is refractory to a PD-1 and/orPD-L1 and/or PD-L2 agent, including, for example, nivolumab(ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB),pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH),MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), Ibrutinib(PHARMACYCLICS/ABBVIE), atezolizumab (TECENTRIQ, GENENTECH), and/orMPDL328OA (ROCHE)-refractory patients. For instance, in embodiments, thesubject is refractory to an anti-CTLA-4 agent, e.g. ipilimumab(YERVOY)-refractory patients (e.g. melanoma patients). Accordingly, inembodiments the present invention provides methods of cancer treatmentthat rescue patients that are non-responsive to various therapies,including monotherapy of one or more immune-modulating agents.

In embodiments, the present methods provide treatment with the chimericprotein in a patient who is refractory to an additional agent, such“additional agents” being described elsewhere herein, inclusive, withoutlimitation, of the various chemotherapeutic agents described herein.

In embodiments, the chimeric proteins are used to treat, control orprevent one or more inflammatory diseases or conditions. Non-limitingexamples of inflammatory diseases include acne vulgaris, acuteinflammation, allergic rhinitis, asthma, atherosclerosis, atopicdermatitis, autoimmune disease, autoinflammatory diseases, autosomalrecessive spastic ataxia, bronchiectasis, celiac disease, chroniccholecystitis, chronic inflammation, chronic prostatitis, colitis,diverticulitis, familial eosinophilia (fe), glomerulonephritis, glycerolkinase deficiency, hidradenitis suppurativa, hypersensitivities,inflammation, inflammatory bowel diseases, inflammatory pelvic disease,interstitial cystitis, laryngeal inflammatory disease, Leigh syndrome,lichen planus, mast cell activation syndrome, mastocytosis, ocularinflammatory disease, otitis, pain, pelvic inflammatory disease,reperfusion injury, respiratory disease, restenosis, rheumatic fever,rheumatoid arthritis, rhinitis, sarcoidosis, septic shock, silicosis andother pneumoconioses, transplant rejection, tuberculosis, andvasculitis.

In various embodiments, the inflammatory disease is an autoimmunedisease or condition, such as multiple sclerosis, diabetes mellitus,lupus, celiac disease, Crohn's disease, ulcerative colitis,Guillain-Barre syndrome, scleroderms, Goodpasture's syndrome, Wegener'sgranulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primarybiliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis,Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Menier'ssyndrome; transplantation rejection (e.g., prevention of allograftrejection) pernicious anemia, rheumatoid arthritis, systemic lupuserythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus,multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave'sdisease, and other autoimmune diseases.

In aspects, the present chimeric agents are used in methods ofactivating a T cell, e.g. via the extracellular domain of OX40L.

In aspects, the present chimeric agents are used in methods ofpreventing the cellular transmission of an immunosuppressive signal viathe extracellular domain of VSIG8.

Combination Therapies and Conjugation

In embodiments, the invention provides for chimeric proteins and methodsthat further comprise administering an additional agent to a subject. Inembodiments, the invention pertains to co-administration and/orco-formulation. Any of the compositions described herein may beco-formulated and/or co-administered.

In embodiments, any chimeric protein described herein actssynergistically when co-administered with another agent and isadministered at doses that are lower than the doses commonly employedwhen such agents are used as monotherapy. In embodiments, any agentreferenced herein may be used in combination with any of the chimericproteins described herein.

In embodiments, the present chimeric protein comprising theextracellular domain of VSIG8 as described herein is co-administeredwith another chimeric protein. In embodiments, the present chimericprotein comprising the extracellular domain of VISIG8 as describedherein is co-administered with another chimeric protein, for example,one which modulates the adaptive immune response. In embodiments, thepresent chimeric protein comprising the extracellular domain of VSIG8 asdescribed herein is co-administered with a chimeric protein comprisingone or more of CSF1R, CD40L, PD-1, GITRL, 4-1BBL, SIRPα, TIM3, TIGIT,and LIGHT. Without wishing to be bound by theory, it is believed that acombined regimen involving the administration of the present chimericprotein which induces an adaptive immune response and one or morechimeric proteins which induces an innate immune response may providesynergistic effects (e.g., synergistic anti-tumor effects).

Any chimeric protein which induces an innate immune response may beutilized in the present invention. For example, the chimeric protein maybe any of the chimeric proteins disclosed in U.S. 62/464,002 whichinduce an innate immune response. In such embodiments, the chimericprotein comprises a first extracellular domain of a type I transmembraneprotein at or near the N-terminus and a second extracellular domain of atype II transmembrane protein at or near the C-terminus, wherein one ofthe first and second extracellular domains provides an immune inhibitorysignal and one of the first and second extracellular domains provides animmune stimulatory signal as disclosed in U.S. 62/464,002, the entirecontents of which is hereby incorporated by reference. In an exemplaryembodiment, the chimeric protein which induces an innate immune responseis a chimeric protein comprising the extracellular domain of CSF1R atthe N-terminus and the extracellular domain of CD40L at the C-terminus.In embodiments, the chimeric protein which induces an innate immuneresponse is a chimeric protein comprising the extracellular domain ofSIRPα at the N-terminus and the extracellular domain of CD40L at theC-terminus.

In embodiments, the present chimeric protein comprising theextracellular domain of VSIG8 as described herein is administered to apatient who has previously been administered a chimeric protein tostimulate the innate immune response (e.g. a CSF1R-based chimericprotein). For instance, the present chimeric protein comprising theextracellular domain of VSIG8 may be administered after the chimericprotein which stimulates the innate immune response (e.g. a CSF1R-basedchimeric protein), including 1 day later, or 2 days later, or 3 dayslater, or 4 days later, or 5 days later, or 6 days later, or 1 weeklater, or 2 weeks later, or 3 weeks later, or 4 weeks later.

In embodiments, inclusive of, without limitation, cancer applications,the present invention pertains to chemotherapeutic agents as additionalagents. Examples of chemotherapeutic agents include, but are not limitedto, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide;alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(e.g., bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; cally statin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINdoxorubicin (including morpholino- doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as minoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine;

pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine);urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., TAXOL paclitaxel (Bristol-Myers SquibbOncology, Princeton, N.J.), ABRAXANE Cremophor-free, albumin-engineerednanoparticle formulation of paclitaxel (American PharmaceuticalPartners, Schaumberg, 111.), and TAXOTERE doxetaxel (Rhone-PoulencRorer, Antony, France); chloranbucil; GEMZAR gemcitabine; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin,oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16);ifosfamide; mitoxantrone; vincristine; NAVELBINE. vinorelbine;novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda;ibandronate; irinotecan (Camptosar, CPT-11) (including the treatmentregimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitorRFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoicacid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin,including the oxaliplatin treatment regimen (FOLFOX); lapatinib(TYKERB); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib(Tarceva)) and VEGF-A that reduce cell proliferation andpharmaceutically acceptable salts, acids or derivatives of any of theabove. In addition, the methods of treatment can further include the useof radiation. In addition, the methods of treatment can further includethe use of photodynamic therapy.

In embodiments, inclusive of, without limitation, cancer applications,the present additional agent is one or more immune-modulating agentsselected from an agent that blocks, reduces and/or inhibits PD-1 and PD-L1 or PD-L2 and/or the binding of PD-1 with PD-L1 or PD-L2 (by way ofnon-limiting example, one or more of nivolumab (ONO-4538/BMS-936558,MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck),pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOLMYERS SQUIBB), atezolizumab (TECENTRIQ, GENENTECH), MPDL328OA (ROCHE)),an agent that increases and/or stimulates CD137 (4-1BB) and/or thebinding of CD137 (4-1BB) with one or more of 4-1BB ligand (by way ofnon-limiting example, urelumab (BMS-663513 and anti-4-1BB antibody), andan agent that blocks, reduces and/or inhibits the activity of CTLA-4and/or the binding of CTLA-4 with one or more of AP2M1, CD80, CD86,SHP-2, and PPP2R5A and/or the binding of OX40 with OX40L (by way ofnon-limiting example GBR 830 (GLENMARK), MEDI6469 (MEDIMMUNE).

In embodiments, inclusive of, without limitation, infectious diseaseapplications, the present invention pertains to anti-infectives asadditional agents. In embodiments, the anti-infective is an anti-viralagent including, but not limited to, Abacavir, Acyclovir, Adefovir,Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine,Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide,Etravirine, Famciclovir, and Foscarnet. In embodiments, theanti-infective is an anti-bacterial agent including, but not limited to,cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil,cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, andceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin,tequin, avelox, and norflox); tetracycline antibiotics (tetracycline,minocycline, oxytetracycline, and doxycycline); penicillin antibiotics(amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin,vancomycin, and methicillin); monobactam antibiotics (aztreonam); andcarbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, andmeropenem). In embodiments, the anti-infectives include anti-malarialagents (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline,artemether/lumefantrine, atovaquone/proguanil andsulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin,pyrantel pamoate, and albendazole.

In embodiments, inclusive, without limitation, of autoimmuneapplications, the additional agent is an immunosuppressive agent. Inembodiments, the immunosuppressive agent is an anti-inflammatory agentsuch as a steroidal anti-inflammatory agent or a non-steroidalanti-inflammatory agent (NSAID). Steroids, particularly the adrenalcorticosteroids and their synthetic analogues, are well known in theart. Examples of corticosteroids useful in the present inventioninclude, without limitation, hydroxyltriamcinolone, alpha-methyldexamethasone, beta-methyl betamethasone, beclomethasone dipropionate,betamethasone benzoate, betamethasone dipropionate, betamethasonevalerate, clobetasol valerate, desonide, desoxymethasone, dexamethasone,diflorasone diacetate, diflucortolone valerate, fluadrenolone,fluclorolone acetonide, flumethasone pivalate, fluosinolone acetonide,fluocinonide, flucortine butylester, fluocortolone, fluprednidene(fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisoneacetate, hydrocortisone butyrate, methylprednisolone, triamcinoloneacetonide, cortisone, cortodoxone, flucetonide, fludrocortisone,difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel,amcinafide, betamethasone and the balance of its esters,chloroprednisone, clocortelone, clescinolone, dichlorisone,difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone,fluprednisolone, hydrocortisone, meprednisone, paramethasone,prednisolone, prednisone, beclomethasone dipropionate. (NSAIDS) that maybe used in the present invention, include but are not limited to,salicylic acid, acetyl salicylic acid, methyl salicylate, glycolsalicylate, salicylmides, benzyl-2,5-diacetoxybenzoic acid, ibuprofen,fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, andindomethacin. In embodiments, the immunosupressive agent may becytostatics such as alkylating agents, antimetabolites (e.g.,azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g.,basiliximab, daclizumab, and muromonab), anti-immunophilins (e.g.,cyclosporine, tacrolimus, sirolimus), inteferons, opioids, TNF bindingproteins, mycophenolates, and small biological agents (e.g., fingolimod,myriocin).

In embodiments, the chimeric proteins (and/or additional agents)described herein, include derivatives that are modified, i.e., by thecovalent attachment of any type of molecule to the composition such thatcovalent attachment does not prevent the activity of the composition.For example, but not by way of limitation, derivatives includecomposition that have been modified by, inter alia, glycosylation,lipidation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications can be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of turicamycin, etc. Additionally, thederivative can contain one or more non-classical amino acids. Inembodiments, the chimeric proteins (and/or additional agents) describedherein further comprise a cytotoxic agent, comprising, in illustrativeembodiments, a toxin, a chemotherapeutic agent, a radioisotope, and anagent that causes apoptosis or cell death. Such agents may be conjugatedto a composition described herein.

The chimeric proteins (and/or additional agents) described herein maythus be modified post-translationally to add effector moieties such aschemical linkers, detectable moieties such as for example fluorescentdyes, enzymes, substrates, bioluminescent materials, radioactivematerials, and chemiluminescent moieties, or functional moieties such asfor example streptavidin, avidin, biotin, a cytotoxin, a cytotoxicagent, and radioactive materials.

Formulations

The chimeric proteins (and/or additional agents) described herein canpossess a sufficiently basic functional group, which can react with aninorganic or organic acid, or a carboxyl group, which can react with aninorganic or organic base, to form a pharmaceutically acceptable salt. Apharmaceutically acceptable acid addition salt is formed from apharmaceutically acceptable acid, as is well known in the art. Suchsalts include the pharmaceutically acceptable salts listed in, forexample, Journal of Pharmaceutical Science, 66, 2-19 (1977) and TheHandbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H.Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, whichare hereby incorporated by reference in their entirety.

In embodiments, the compositions described herein are in the form of apharmaceutically acceptable salt.

Further, any chimeric protein (and/or additional agents) describedherein can be administered to a subject as a component of a compositionthat comprises a pharmaceutically acceptable carrier or vehicle. Suchcompositions can optionally comprise a suitable amount of apharmaceutically acceptable excipient so as to provide the form forproper administration. Pharmaceutical excipients can be liquids, such aswater and oils, including those of petroleum, animal, vegetable, orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. The pharmaceutical excipients can be, for example,saline, gum acacia, gelatin, starch paste, talc, keratin, colloidalsilica, urea and the like. In addition, auxiliary, stabilizing,thickening, lubricating, and coloring agents can be used. Inembodiments, the pharmaceutically acceptable excipients are sterile whenadministered to a subject. Water is a useful excipient when any agentdescribed herein is administered intravenously. Saline solutions andaqueous dextrose and glycerol solutions can also be employed as liquidexcipients, specifically for injectable solutions. Suitablepharmaceutical excipients also include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Any agent describedherein, if desired, can also comprise minor amounts of wetting oremulsifying agents, or pH buffering agents.

In embodiments, the compositions described herein are suspended in asaline buffer (including, without limitation TBS, PBS, and the like).

In embodiments, the chimeric proteins may by conjugated and/or fusedwith another agent to extend half-life or otherwise improvepharmacodynamic and pharmacokinetic properties. In embodiments, thechimeric proteins may be fused or conjugated with one or more of PEG,XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., humanserum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP,transferrin, and the like. In embodiments, each of the individualchimeric proteins is fused to one or more of the agents described inBioDrugs (2015) 29:215-239, the entire contents of which are herebyincorporated by reference.

Administration, Dosing, and Treatment Regimens

The present invention includes the described chimeric protein (and/oradditional agents) in various formulations. Any chimeric protein (and/oradditional agents) described herein can take the form of solutions,suspensions, emulsion, drops, tablets, pills, pellets, capsules,capsules containing liquids, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, or any otherform suitable for use. DNA or RNA constructs encoding the proteinsequences may also be used. In embodiments, the composition is in theform of a capsule (see, e.g., U.S. Pat. No. 5,698,155). Other examplesof suitable pharmaceutical excipients are described in Remington'sPharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed.1995), incorporated herein by reference.

Where necessary, the formulations comprising the chimeric protein(and/or additional agents) can also include a solubilizing agent. Also,the agents can be delivered with a suitable vehicle or delivery deviceas known in the art. Combination therapies outlined herein can beco-delivered in a single delivery vehicle or delivery device.Compositions for administration can optionally include a localanesthetic such as, for example, lignocaine to lessen pain at the siteof the injection.

The formulations comprising the chimeric protein (and/or additionalagents) of the present invention may conveniently be presented in unitdosage forms and may be prepared by any of the methods well known in theart of pharmacy. Such methods generally include the step of bringing thetherapeutic agents into association with a carrier, which constitutesone or more accessory ingredients. Typically, the formulations areprepared by uniformly and intimately bringing the therapeutic agent intoassociation with a liquid carrier, a finely divided solid carrier, orboth, and then, if necessary, shaping the product into dosage forms ofthe desired formulation (e.g., wet or dry granulation, powder blends,etc., followed by tableting using conventional methods known in the art)

In embodiments, any chimeric protein (and/or additional agents)described herein is formulated in accordance with routine procedures asa composition adapted for a mode of administration described herein.

Routes of administration include, for example: intradermal,intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, oral, sublingual, intranasal, intracerebral,intravaginal, transdermal, rectally, by inhalation, or topically,particularly to the ears, nose, eyes, or skin. In embodiments, theadministering is effected orally or by parenteral injection. In someinstances, administration results in the release of any agent describedherein into the bloodstream, or alternatively, the agent is administereddirectly to the site of active disease.

Any chimeric protein (and/or additional agents) described herein can beadministered orally. Such chimeric proteins (and/or additional agents)can also be administered by any other convenient route, for example, byintravenous infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and can be administered together with anotherbiologically active agent. Administration can be systemic or local.Various delivery systems are known, e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, etc., and can be used toadminister.

In specific embodiments, it may be desirable to administer locally tothe area in need of treatment. In embodiments, for instance in thetreatment of cancer, the chimeric protein (and/or additional agents) areadministered in the tumor microenvironment (e.g. cells, molecules,extracellular matrix and/or blood vessels that surround and/or feed atumor cell, inclusive of, for example, tumor vasculature;tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelialprogenitor cells (EPC); cancer-associated fibroblasts; pericytes; otherstromal cells; components of the extracellular matrix (ECM); dendriticcells; antigen presenting cells; T-cells; regulatory T cells;macrophages; neutrophils; and other immune cells located proximal to atumor) or lymph node and/or targeted to the tumor microenvironment orlymph node. In embodiments, for instance in the treatment of cancer, thechimeric protein (and/or additional agents) are administeredintratumorally.

In embodiments, the present chimeric protein allows for a dual effectthat provides less side effects than are seen in conventionalimmunotherapy (e.g. treatments with one or more of OPDIVO, KEYTRUDA,YERVOY, and TECENTRIQ). For example, the present chimeric proteinsreduce or prevent commonly observed immune-related adverse events thataffect various tissues and organs including the skin, thegastrointestinal tract, the kidneys, peripheral and central nervoussystem, liver, lymph nodes, eyes, pancreas, and the endocrine system;such as hypophysitis, colitis, hepatitis, pneumonitis, rash, andrheumatic disease. Further, the present local administration, e.g.intratumorally, obviate adverse event seen with standard systemicadministration, e.g. IV infusions, as are used with conventionalimmunotherapy (e.g. treatments with one or more of OPDIVO, KEYTRUDA,YERVOY, and TECENTRIQ).

Dosage forms suitable for parenteral administration (e.g. intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g. lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use. They may contain, for example, suspending ordispersing agents known in the art.

The dosage of any chimeric protein (and/or additional agents) describedherein as well as the dosing schedule can depend on various parameters,including, but not limited to, the disease being treated, the subject'sgeneral health, and the administering physician's discretion. Anychimeric protein described herein, can be administered prior to (e.g., 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of anadditional agent, to a subject in need thereof. In embodiments anychimeric protein and additional agent described herein are administered1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hourapart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart,3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hoursapart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11hours to 12 hours apart, 1 day apart, 2 days apart, 3 days part, 4 daysapart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeksapart, or 4 weeks apart.

In embodiments, the present invention relates to the co-administrationof the present chimeric protein comprising the extracellular domain ofVSIG8 and another chimeric protein which induces an innate immuneresponse. In such embodiments, the present chimeric protein may beadministered before, concurrently with, or subsequent to administrationof the chimeric protein which induces an innate immune response. Forexample, the chimeric proteins may be administered 1 minute apart, 10minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hoursapart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hoursto 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hoursapart, 1 day apart, 2 days apart, 3 days part, 4 days apart, 5 daysapart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4weeks apart. In an exemplary embodiment, the present chimeric proteincomprising the extracellular domain of VSIG8 and the chimeric proteinwhich induces an innate immune response are administered 1 week apart,or administered on alternate weeks (i.e., administration of the chimericprotein inducing an innate immune response is followed 1 week later withadministration of the present chimeric protein comprising theextracellular domain of VSIG8 and so forth).

The dosage of any chimeric protein (and/or additional agents) describedherein can depend on several factors including the severity of thecondition, whether the condition is to be treated or prevented, and theage, weight, and health of the subject to be treated. Additionally,pharmacogenomic (the effect of genotype on the pharmacokinetic,pharmacodynamic or efficacy profile of a therapeutic) information abouta particular subject may affect dosage used. Furthermore, the exactindividual dosages can be adjusted somewhat depending on a variety offactors, including the specific combination of the agents beingadministered, the time of administration, the route of administration,the nature of the formulation, the rate of excretion, the particulardisease being treated, the severity of the disorder, and the anatomicallocation of the disorder. Some variations in the dosage can be expected.

For administration of any chimeric protein (and/or additional agents)described herein by parenteral injection, the dosage may be about 0.1 mgto about 250 mg per day, about 1 mg to about 20 mg per day, or about 3mg to about 5 mg per day. Generally, when orally or parenterallyadministered, the dosage of any agent described herein may be about 0.1mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, orabout 0.5 mg to about 5 mg per day, or about 200 to about 1,200 mg perday (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about1,100 mg, about 1,200 mg per day).

In embodiments, administration of the chimeric protein (and/oradditional agents) described herein is by parenteral injection at adosage of about 0.1 mg to about 1500 mg per treatment, or about 0.5 mgto about 10 mg per treatment, or about 0.5 mg to about 5 mg pertreatment, or about 200 to about 1,200 mg per treatment (e.g., about 200mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about1,200 mg per treatment).

In embodiments, a suitable dosage of the chimeric protein (and/oradditional agents) is in a range of about 0.01 mg/kg to about 100 mg/kgof body weight ,or about 0.01 mg/kg to about 10 mg/kg of body weight ofthe subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg,about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg,about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kgbody weight, inclusive of all values and ranges therebetween.

In embodiments, delivery can be in a vesicle, in particular a liposome(see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes inthe Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler(eds.), Liss, New York, pp. 353-365 (1989).

Any chimeric protein (and/or additional agents) described herein can beadministered by controlled-release or sustained-release means or bydelivery devices that are well known to those of ordinary skill in theart. Examples include, but are not limited to, those described in U.S.Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719;5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476;5,354,556; and 5,733,556, each of which is incorporated herein byreference in its entirety. Such dosage forms can be useful for providingcontrolled- or sustained-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Controlled-or sustained-release of an active ingredient can be stimulated byvarious conditions, including but not limited to, changes in pH, changesin temperature, stimulation by an appropriate wavelength of light,concentration or availability of enzymes, concentration or availabilityof water, or other physiological conditions or compounds.

In embodiments, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fl. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61;see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann.Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

In embodiments, a controlled-release system can be placed in proximityof the target area to be treated, thus requiring only a fraction of thesystemic dose (see, e.g., Goodson, in Medical Applications of ControlledRelease, supra, vol. 2, pp. 115-138 (1984)). Other controlled-releasesystems discussed in the review by Langer, 1990, Science 249:1527-1533)may be used.

Administration of any chimeric protein (and/or additional agents)described herein can, independently, be one to four times daily or oneto four times per month or one to six times per year or once every two,three, four or five years. Administration can be for the duration of oneday or one month, two months, three months, six months, one year, twoyears, three years, and may even be for the life of the subject.

The dosage regimen utilizing any chimeric protein (and/or additionalagents) described herein can be selected in accordance with a variety offactors including type, species, age, weight, sex and medical conditionof the subject; the severity of the condition to be treated; the routeof administration; the renal or hepatic function of the subject; thepharmacogenomic makeup of the individual; and the specific compound ofthe invention employed. Any chimeric protein (and/or additional agents)described herein can be administered in a single daily dose, or thetotal daily dosage can be administered in divided doses of two, three orfour times daily. Furthermore, any chimeric protein (and/or additionalagents) described herein can be administered continuously rather thanintermittently throughout the dosage regimen.

Cells and Nucleic Acids

In embodiments, the present invention provides an expression vector,comprising a nucleic acid encoding the chimeric protein describedherein. In embodiments, the expression vector comprises DNA or RNA. Inembodiments, the expression vector is a mammalian expression vector.

Both prokaryotic and eukaryotic vectors can be used for expression ofthe chimeric protein. Prokaryotic vectors include constructs based on E.coli sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512-538).Non-limiting examples of regulatory regions that can be used forexpression in E. coli include lac, trp, Ipp, phoA, recA, tac, T3, T7 andλP_(L). Non-limiting examples of prokaryotic expression vectors mayinclude the λgt vector series such as λgt11 (Huynh et al., in “DNACloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover,ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studieret al., Methods Enzymol 1990, 185:60-89). Prokaryotic host-vectorsystems cannot perform much of the post-translational processing ofmammalian cells, however. Thus, eukaryotic host-vector systems may beparticularly useful. A variety of regulatory regions can be used forexpression of the chimeric proteins in mammalian host cells. Forexample, the SV40 early and late promoters, the cytomegalovirus (CMV)immediate early promoter, and the Rous sarcoma virus long terminalrepeat (RSV-LTR) promoter can be used. Inducible promoters that may beuseful in mammalian cells include, without limitation, promotersassociated with the metallothionein II gene, mouse mammary tumor virusglucocorticoid responsive long terminal repeats (MMTV-LTR), theβ-interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75).Heat shock promoters or stress promoters also may be advantageous fordriving expression of the chimeric proteins in recombinant host cells.

In embodiments, expression vectors of the invention comprise a nucleicacid encoding the chimeric proteins (and/or additional agents), or acomplement thereof, operably linked to an expression control region, orcomplement thereof, that is functional in a mammalian cell. Theexpression control region is capable of driving expression of theoperably linked blocking and/or stimulating agent encoding nucleic acidsuch that the blocking and/or stimulating agent is produced in a humancell transformed with the expression vector.

Expression control regions are regulatory polynucleotides (sometimesreferred to herein as elements), such as promoters and enhancers, thatinfluence expression of an operably linked nucleic acid. An expressioncontrol region of an expression vector of the invention is capable ofexpressing operably linked encoding nucleic acid in a human cell. Inembodiments, the cell is a tumor cell. In embodiments, the cell is anon-tumor cell. In embodiments, the expression control region confersregulatable expression to an operably linked nucleic acid. A signal(sometimes referred to as a stimulus) can increase or decreaseexpression of a nucleic acid operably linked to such an expressioncontrol region. Such expression control regions that increase expressionin response to a signal are often referred to as inducible. Suchexpression control regions that decrease expression in response to asignal are often referred to as repressible. Typically, the amount ofincrease or decrease conferred by such elements is proportional to theamount of signal present; the greater the amount of signal, the greaterthe increase or decrease in expression.

In embodiments, the present invention contemplates the use of induciblepromoters capable of effecting high level of expression transiently inresponse to a cue. For example, when in the proximity of a tumor cell, acell transformed with an expression vector for the chimeric protein(and/or additional agents) comprising such an expression controlsequence is induced to transiently produce a high level of the agent byexposing the transformed cell to an appropriate cue. Illustrativeinducible expression control regions include those comprising aninducible promoter that is stimulated with a cue such as a smallmolecule chemical compound. Particular examples can be found, forexample, in U.S. Pat. Nos. 5,989,910, 5,935,934, 6,015,709, and6,004,941, each of which is incorporated herein by reference in itsentirety.

Expression control regions and locus control regions include full-lengthpromoter sequences, such as native promoter and enhancer elements, aswell as subsequences or polynucleotide variants which retain all or partof full-length or non-variant function. As used herein, the term“functional” and grammatical variants thereof, when used in reference toa nucleic acid sequence, subsequence or fragment, means that thesequence has one or more functions of native nucleic acid sequence(e.g., non-variant or unmodified sequence).

As used herein, “operable linkage” refers to a physical juxtaposition ofthe components so described as to permit them to function in theirintended manner. In the example of an expression control element inoperable linkage with a nucleic acid, the relationship is such that thecontrol element modulates expression of the nucleic acid. Typically, anexpression control region that modulates transcription is juxtaposednear the 5′ end of the transcribed nucleic acid (i.e., “upstream”).Expression control regions can also be located at the 3′ end of thetranscribed sequence (i.e., “downstream”) or within the transcript(e.g., in an intron). Expression control elements can be located at adistance away from the transcribed sequence (e.g., 100 to 500, 500 to1000, 2000 to 5000, or more nucleotides from the nucleic acid). Aspecific example of an expression control element is a promoter, whichis usually located 5′ of the transcribed sequence. Another example of anexpression control element is an enhancer, which can be located 5′ or 3′of the transcribed sequence, or within the transcribed sequence.

Expression systems functional in human cells are well known in the art,and include viral systems. Generally, a promoter functional in a humancell is any DNA sequence capable of binding mammalian RNA polymerase andinitiating the downstream (3′) transcription of a coding sequence intomRNA. A promoter will have a transcription initiating region, which isusually placed proximal to the 5′ end of the coding sequence, andtypically a TATA box located 25-30 base pairs upstream of thetranscription initiation site.

The TATA box is thought to direct RNA polymerase II to begin RNAsynthesis at the correct site. A promoter will also typically contain anupstream promoter element (enhancer element), typically located within100 to 200 base pairs upstream of the TATA box. An upstream promoterelement determines the rate at which transcription is initiated and canact in either orientation. Of particular use as promoters are thepromoters from mammalian viral genes, since the viral genes are oftenhighly expressed and have a broad host range. Examples include the SV40early promoter, mouse mammary tumor virus LTR promoter, adenovirus majorlate promoter, herpes simplex virus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenylation signals includethose derived from SV40. Introns may also be included in expressionconstructs.

There are a variety of techniques available for introducing nucleicacids into viable cells. Techniques suitable for the transfer of nucleicacid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, polymer-based systems,DEAE-dextran, viral transduction, the calcium phosphate precipitationmethod, etc. For in vivo gene transfer, a number of techniques andreagents may also be used, including liposomes; natural polymer-baseddelivery vehicles, such as chitosan and gelatin; viral vectors are alsosuitable for in vivo transduction. In some situations, it is desirableto provide a targeting agent, such as an antibody or ligand specific fora tumor cell surface membrane protein. Where liposomes are employed,proteins which bind to a cell surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake, e.g.,capsid proteins or fragments thereof tropic for a particular cell type,antibodies for proteins which undergo internalization in cycling,proteins that target intracellular localization and enhanceintracellular half-life. The technique of receptor-mediated endocytosisis described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414(1990).

Where appropriate, gene delivery agents such as, e.g., integrationsequences can also be employed. Numerous integration sequences are knownin the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406,1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell,122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra etal., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These includerecombinases and transposases. Examples include Cre (Sternberg andHamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247,543-545, 1974), Flp (Broach, et al., Cell, 29:227-234, 1982), R(Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see,e.g., Groth et al., J. Mol. Biol. 335:667-678, 2004), sleeping beauty,transposases of the mariner family (Plasterk et al., supra), andcomponents for integrating viruses such as AAV, retroviruses, andantiviruses having components that provide for virus integration such asthe LTR sequences of retroviruses or lentivirus and the ITR sequences ofAAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). Inaddition, direct and targeted genetic integration strategies may be usedto insert nucleic acid sequences encoding the chimeric proteinsincluding CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editingtechnologies.

In one aspect, the invention provides expression vectors for theexpression of the chimeric proteins (and/or additional agents) that areviral vectors. Many viral vectors useful for gene therapy are known(see, e.g., Lundstrom, Trends Biotechnol., 21:1 17, 122, 2003.Illustrative viral vectors include those selected from Antiviruses (LV),retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV),and α viruses, though other viral vectors may also be used. For in vivouses, viral vectors that do not integrate into the host genome aresuitable for use, such as α viruses and adenoviruses. Illustrative typesof α viruses include Sindbis virus, Venezuelan equine encephalitis (VEE)virus, and Semliki Forest virus (SFV). For in vitro uses, viral vectorsthat integrate into the host genome are suitable, such as retroviruses,AAV, and Antiviruses. In embodiments, the invention provides methods oftransducing a human cell in vivo, comprising contacting a solid tumor invivo with a viral vector of the invention.

In embodiments, the present invention provides a host cell, comprisingthe expression vector comprising the chimeric protein described herein.

Expression vectors can be introduced into host cells for producing thepresent chimeric proteins. Cells may be cultured in vitro or geneticallyengineered, for example. Useful mammalian host cells include, withoutlimitation, cells derived from humans, monkeys, and rodents (see, forexample, Kriegler in “Gene Transfer and Expression: A LaboratoryManual,” 1990, New York, Freeman & Co.). These include monkey kidneycell lines transformed by SV40 (e.g., COS-7, ATCC CRL 1651); humanembryonic kidney lines (e.g., 293, 293-EBNA, or 293 cells subcloned forgrowth in suspension culture, Graham et al., J Gen Virol 1977, 36:59);baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamsterovary-cells-DHFR (e.g., CHO, Urlaub and Chasin, Proc Natl Aced Sci USA1980, 77:4216); DG44 CHO cells, CHO-K1 cells, mouse sertoli cells(Mather, Biol Reprod 1980, 23:243-251); mouse fibroblast cells (e.g.,NIH-3T3), monkey kidney cells (e.g., CV1 ATCC CCL 70); African greenmonkey kidney cells. (e.g., VERO-76, ATCC CRL-1587); human cervicalcarcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g.,MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A, ATCC CRL1442); human lung cells (e.g., W138, ATCC CCL 75); human liver cells(e.g., Hep G2, HB 8065); and mouse mammary tumor cells (e.g., MMT060562, ATCC CCL51). Illustrative cancer cell types for expressing thechimeric proteins described herein include mouse fibroblast cell line,NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytomacell line, P815, mouse lymphoma cell line, EL4 and its ovalbumintransfectant, E.G7, mouse melanoma cell line, B16F10, mouse fibrosarcomacell line, MC57, and human small cell lung carcinoma cell lines, SCLC#2and SCLC#7.

Host cells can be obtained from normal or affected subjects, includinghealthy humans, cancer patients, and patients with an infectiousdisease, private laboratory deposits, public culture collections such asthe American Type Culture Collection, or from commercial suppliers.

Cells that can be used for production of the present chimeric proteinsin vitro, ex vivo, and/or in vivo include, without limitation,epithelial cells, endothelial cells, keratinocytes, fibroblasts, musclecells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes,monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,granulocytes; various stem or progenitor cells, in particularhematopoietic stem or progenitor cells (e.g., as obtained from bonemarrow), umbilical cord blood, peripheral blood, fetal liver, etc. Thechoice of cell type depends on the type of tumor or infectious diseasebeing treated or prevented, and can be determined by one of skill in theart.

Subjects and/or Animals

In embodiments, the subject and/or animal is a mammal, e.g., a human,mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, ornon-human primate, such as a monkey, chimpanzee, or baboon. Inembodiments, the subject and/or animal is a non-mammal, such, forexample, a zebrafish. In embodiments, the subject and/or animal maycomprise fluorescently-tagged cells (with e.g. GFP). In embodiments, thesubject and/or animal is a transgenic animal comprising a fluorescentcell.

In embodiments, the subject and/or animal is a human. In embodiments,the human is a pediatric human. In embodiments, the human is an adulthuman. In embodiments, the human is a geriatric human. In embodiments,the human may be referred to as a patient.

In certain embodiments, the human has an age in a range of from about 0months to about 6 months old, from about 6 to about 12 months old, fromabout 6 to about 18 months old, from about 18 to about 36 months old,from about 1 to about 5 years old, from about 5 to about 10 years old,from about 10 to about 15 years old, from about 15 to about 20 yearsold, from about 20 to about 25 years old, from about 25 to about 30years old, from about 30 to about 35 years old, from about 35 to about40 years old, from about 40 to about 45 years old, from about 45 toabout 50 years old, from about 50 to about 55 years old, from about 55to about 60 years old, from about 60 to about 65 years old, from about65 to about 70 years old, from about 70 to about 75 years old, fromabout 75 to about 80 years old, from about 80 to about 85 years old,from about 85 to about 90 years old, from about 90 to about 95 years oldor from about 95 to about 100 years old.

In embodiments, the subject is a non-human animal, and therefore theinvention pertains to veterinary use. In a specific embodiment, thenon-human animal is a household pet. In another specific embodiment, thenon-human animal is a livestock animal.

Kits

The invention provides kits that can simplify the administration of anyagent described herein. An illustrative kit of the invention comprisesany composition described herein in unit dosage form. In embodiments,the unit dosage form is a container, such as a pre-filled syringe, whichcan be sterile, containing any agent described herein and apharmaceutically acceptable carrier, diluent, excipient, or vehicle. Thekit can further comprise a label or printed instructions instructing theuse of any agent described herein. The kit may also include a lidspeculum, topical anesthetic, and a cleaning agent for theadministration location. The kit can also further comprise one or moreadditional agent described herein. In embodiments, the kit comprises acontainer containing an effective amount of a composition of theinvention and an effective amount of another composition, such thosedescribed herein.

Any aspect or embodiment described herein can be combined with any otheraspect or embodiment as disclosed herein.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1: In Silico Predicted Secondary Structure of HumanVSIG8-Fc-OX40L Chimeric Protein

An in silico structure prediction of a human VSIG8-Fc-OX40L chimericprotein having 603 amino acid residues, with a p-value 5.36×10⁻¹⁶. Themolecular weight of the monomeric protein was predicted to beapproximately 68.1 kDa. A structure of the chimeric protein is providedin FIG. 1A. FIG. 1B shows a synapse that has formed by a chimericprotein between a tumor cell and a T cell.

Secondary structure prediction of the entire sequence of the chimericprotein showed that the protein has the composition of 0% α-helix (H),58% β-sheet (E), and 41% coil (C). The GDT (global distance test) anduGDT (un-normalized GDT) for the absolute global quality were alsocalculated for the chimeric protein to give an overall uGDT(GDT) of 362(60). The three-state prediction for solvent accessibility of theprotein residues were 33% exposed (E), 47% intermediate (M), and 19%buried (B).

Example 2: Characterization of Human VSIG8-Fc-OX40L Chimeric Protein

A human VSIG8-Fc-OX40L chimeric protein was constructed as describedabove in the Detailed Description and in U.S. 62/464,002, the contentsof which are hereby incorporated by reference in its entirety. Thechimeric protein was characterized by performing a Western blot analysisagainst each domain of the chimeric protein, i.e., via anti-VSIG8,anti-Fc, and anti-OX40L antibodies.

The Western blots indicated the presence of a dominant dimeric band inthe non-reduced lanes (no β-mercaptoethanol or PNGase; FIG. 2, lane 2 ineach blot), which was reduced to a glycosylated monomeric band in thepresence of the reducing agent, β-mercaptoethanol (FIG. 2, lane 3 ineach blot). As shown in FIG. 2, lane 4 in each blot, the chimericprotein ran as a monomer at the predicted molecular weight ofapproximately 68.1 kDa in the presence of both a reducing agent(β-mercaptoethanol) and the endoglycosidase Peptide:N-Glycosidase(PNGase).

Example 3: Characterization of the Binding Affinity of the DifferentDomains of the Human VSIG8-Fc-OX40L Chimeric Protein using ELISA

Enzyme-Linked Immunosorbent assay (ELISA) assays were developed todemonstrate the binding affinity of the different domains of humanVSIG8-Fc-OX40L to respective hlgG or recombinant OX40L. Specifically,the Fc portion of the chimeric protein was detected by capturing to aplate-bound human IgG and detecting via an HRP-conjugated anti-human IgGantibody (left panel of FIG. 3). The OX40 domain of the chimeric proteinwas detected by capturing to a plate-bound recombinant human OX40protein and detecting via an anti-OX40-specific antibody (right panel ofFIG. 3). It was observed that in ELISA assays, using the central Fcregion to detect chimeric proteins tended to underestimate the actualprotein content in a sample. Therefore, low level of the hVSIG8-Fc-OX40Lchimeric protein was detected compared to standard in this assay.

Example 4: Characterization of the In Vitro Cell Binding Affinity ofHuman VSIG8-Fc-OX40L Chimeric Protein

Cell binding assays were performed to demonstrate the binding affinityof the different domains of the human VSIG8-Fc-OX40L chimeric proteintowards their respective binding partners on the surface of a mammaliancell membrane.

For cell binding assays, immortalized cell lines were engineered tostably express human OX40 (Jurkat/hOX40). Increasing concentrations ofthe VSIG8-Fc-OX40L chimeric protein were incubated with theover-expressing (Jurkat/hOX40) cell line for two hours. Cells werecollected, washed, and stained with antibodies for the detection ofchimeric protein binding by flow cytometry.

As shown in FIG. 4, the VSIG8-Fc-OX40L chimeric protein bound to OX40present on the cell surface in a concentration-dependent manner and withlow nM affinity. Specifically, as shown in FIG. 4, the cell bindingassay demonstrated that VSIG8-Fc-OX40L binds to OX40 with an affinity ofabout 30 nM (according to the EC₅₀ calculation).

Example 5: Characterization of the Binding Affinity of HumanVSIG8-Fc-OX40L Chimeric Protein by Surface Plasmon Resonance (SPR)

The binding affinity of the OX40L domain of the human VSIG8-Fc-OX40Lchimeric protein was measured by the surface plasmon resonance (SPR)using the BioRad ProteOn XPR 360 system. Specifically, the affinity ofthe chimeric protein for human OX40 was determined and compared to arecombinant control protein and to tavolixumab (an anti-human OX40antibody); the results are shown in FIG. 5 and the below table:

Conc. Kon Kdis KD Sample ID Ligand (nM) (1/Ms) (1/s) (nM) Fc-OX40LhOX40-His 222.2 4.17E+04 2.54E−03 61 Tavolixumab hOX40-His 66.7 3.32E+055.03E−05 0.152 VSIG8-Fc-OX40L hOX40-His 117.6 3.31E+05 2.54E−04 0.767

It was determined that the VSIG8-Fc-OX40L chimeric protein binds tohOX40 with high affinity. In particular, it was noted that the off-ratesof the hVSIG8-Fc-OX40L chimeric protein are much slower than the controlFc-OX40L protein. For example, the off-rate of the chimeric protein fromOX40L was 10 fold slower than the Fc-OX40L protein.

Example 6. Induction of OX40 Signaling In Vitro

Human OX40 activation leads to induction of a signaling cascade whichinvolves both NF-κB and NIK activation. FIG. 6 shows example data froman in vitro NF-κB/NIK signaling assay using the human VSIG8-Fc-OX40Lchimeric protein. U2OS cells from the DiscoverX NIK signaling assay werecultured with a titration of either a commercially-availablesingle-sided hFc-OX40L, an anti-OX40L antibody (Oxelumab), an anti-OX40antibody (Tavolixumab), or the human VSIG8-Fc-OX40L chimeric protein.The relative luciferase units (RLU) indicate the relative strength ofNF-κB/NIK signaling activated following treatment with the indicatedregimens. VSIG8-Fc-OX40L is shown to have strongly activated signalingvia NF-κB and NIK and to a greater degree than any of the otherindicated regimen.

Example 7: Functional Activity of Human VSIG8-Fc-OX40L Chimeric Proteinin a Superantigen Cytokine Release Assay

Another functional assay conducted to characterize the functionalactivity of human VSIG8-Fc-OX40L chimeric protein is the superantigencytokine release assay. In this assay, 200 ng/ml of staphylococcusenterotoxin B (SEB) were used to activate human peripheral bloodleukocytes in the presence of various concentrations test agents, i.e.,single-sided human Fc-OX40L, commercially-available, single-sidedVSIG8-Fc, a combination of the two single-sided molecules, or the humanVSIG8-Fc-OX40L chimeric protein. Three days later, supernatants wereassessed using ELISAs specific to human IL2.

As shown in FIG. 7, the quantity of IL-2 secreted into the culturesupernatant was monitored as a functional readout of the ability of testagents to either block suppressive signaling events or co-stimulateimmune activating signals. At concentrations above 0.5 nm, theVSIG8-Fc-OX40L chimeric protein induced secretion of IL2 at higherlevels than any other test agents or combinations of agents and at anyconcentration (up to 50 nM). Media and IgG controls were used. Together,these results suggest that hVSIG8-Fc-OX40L chimeric protein functionallyactivated primary human leukocytes cells in vitro.

Example 8: Characterization of the Binding Affinity of the DifferentDomains of the VSIG8-Fc-OX40L Chimeric Protein Using ELISA

ELISA (enzyme-linked immunosorbent assay) assays were developed todemonstrate the binding affinity of the different domains of the murineVSIG8-Fc-OX40L to their respective binding partners (i.e., VISTA, humanIgG (hIgG), or OX40). Specifically, the VSIG8 domain of themVSIG8-Fc-OX40L chimeric protein was detected by capturing to aplate-bound recombinant murine VISTA protein and detecting using anOX40L specific antibody. The Fc portion of the chimeric protein wasdetected by capturing to a plate-bound hIgG and detecting via an HRPconjugated anti-hlgG antibody. The OX40L domain of the chimeric proteinwas detected by capturing to a plate-bound recombinant murine OX40protein and detecting via an OX40L specific antibody.

As shown in FIG. 8, the different domains of the mVSIG8-Fc-OX40Lchimeric protein effectively interacted with their respective bindingpartners with high affinity. Nevertheless, it was observed that in ELISAassays, using the central Fc region to detect chimeric proteins tendedto underestimate the actual protein content in a sample. Therefore, lowlevel of the VSIG8-Fc-OX40L chimeric protein was detected compared tostandard in this assay. The band sizes confirm the predicted monomericmolecular weight of approximately 68.1 kDa and suggest that the chimericprotein's native state is as a glycosylated dimer.

Example 9: Characterization of Murine VSIG8-Fc-OX40L Chimeric Protein

A murine VSIG8-Fc-OX40L chimeric protein was constructed as describedabove in the Detailed Description and in U.S. 62/464,002, the contentsof which are hereby incorporated by reference in its entirety.

FIG. 9A to FIG. 9C show ELISA assays demonstrating binding affinity ofthe different domains of murine VSIG8-Fc-OX40L chimeric protein fortheir respective binding partners. Specifically, the VISIG8 domain ofthe mVSIG8-Fc-OX40L chimeric protein was detected by capturing to aplate-bound recombinant murine VISTA protein (the binding partner forVSIG8) and detecting via a HRP-conjugated anti-mouse IgG antibody (FIG.9A). A commercially-available mVSIG8-Fc standard is unavailable;therefore, no standard curve was generated. The Fc portion of thechimeric protein was detected by capturing to a plate-bound mouse IgGand detecting via an HRP-conjugated anti-mouse IgG antibody (FIG. 9B).The OX40L domain of the chimeric protein was detected by capturing to aplate-bound recombinant mouse OX40 protein and detecting via anOX40L-specific antibody (FIG. 9C). FIG. 9A to FIG. 9C show that thedifferent domains of the mVSIG8-Fc-OX40L chimeric protein effectivelyinteracted with their respective binding partners with high affinity.Nevertheless, it was observed that in ELISA assays, using the central Fcregion to detect chimeric proteins tended to underestimate the actualprotein content in a sample. Therefore, low level of the mVSIG8-Fc-OX40Lchimeric protein was detected compared to standard in this assay.

Example 10: Characterization of the In Vitro Cell Binding Affinity ofthe VSIG8-Fc-OX40L Chimeric Protein

Cell binding assays were performed to demonstrate the binding affinityof the different domains of the murine VSIG8-Fc-OX40L chimeric proteintowards their respective binding partners on the surface of a mammaliancell membrane.

For the cell binding assays, immortalized cell lines were engineered tostably express murine VISTA (EL4-mVISTA) and the murine receptor OX40(CHOK1-mOX40). Increasing concentrations of mVSIG8-Fc-OX40L wereincubated with each parental (control) and over-expressing cell linesfor two hours. Cells were collected, washed, and stained with antibodiesfor the detection of chimeric protein binding by flow cytometry.

As shown in FIG. 10A and FIG. 10B, the mVSIG8-Fc-OX40L chimeric proteinbound to mVISTA and mOX40 present on the cell surface in aconcentration-dependent manner and with low nM affinity. Specifically,as shown in FIG. 10A, the EL4-parental cell line (bottom curve) was notresponsive to increasing concentrations of the mVSIG8-Fc-OX40L chimericprotein as it did not overexpress VISTA. In comparison, the EL4-mVISTAcell line (top curve), which was engineered to overexpress mVISTA, boundto mVSIG8-Fc-OX40L in a concentration-dependent manner. Similarly, asshown in FIG. 10B, the CHOK1-parental cell line (bottom curve) was notresponsive to increasing concentrations of the mVSIG8-Fc-OX40L chimericprotein as it did not overexpress mOX40. In contrast, the CHOK1-mOX40cell line (top curve), which was engineered to overexpress mOX40, boundto mVSIG8-Fc-OX40L in a concentration-dependent manner. The cell bindingassay also indicated that mVSIG8-Fc-OX40L bound to mOX40 with anaffinity of 16 nM, and to mVISTA with an affinity of 56 nM (according tothe EC₅₀ calculation).

Example 11: Characterization of the Binding Affinity of MurineVSIG8-Fc-OX40L Chimeric Protein by Surface Plasmon Resonance (SPR)

The binding affinity of the OX40L domain of the murine VSIG8-Fc-OX40Lchimeric protein was measured by the surface plasmon resonance (SPR)using the BioRad ProteOn XPR 360 system. Specifically, the affinity ofthe chimeric protein for murine OX40 was determined and compared to acommercially-available recombinant control protein (mFc-OX40L); theresults are shown in FIG. 11. It was determined that the mVSIG8-Fc-OX40Lchimeric protein binds to mOX40 with high affinity.

Example 12: Induction of OX40 Signaling In Vitro

Murine OX40 activation leads to induction of a signaling cascade whichinvolves both NF-κB and NIK activation. FIG. 12 shows example data froman in vitro NF-κB/NIK signaling assay using the murine VSIG8-Fc-OX40Lchimeric protein. U2OS cells from the DiscoverX NIK signaling assay werecultured with a titration of either an irrelevant protein, acommercially-available single-sided hFc-OX40L, or the mVSIG8-Fc-OX40Lchimeric protein. The relative luciferase units (RLU) indicate therelative strength of NF-κB/NIK signaling activated following treatmentwith the indicated regimens. VSIG8-Fc-OX40L is shown to have stronglyactivated signaling via NF-κB and NIK and to a greater degree than anyof the other indicated regimen.

Example 13: Functional Assays of the VSIG8-Fc-OX40L Chimeric Protein

In vivo functional assays were performed to demonstrate the functionalactivity of the murine VSIG8-Fc-OX40L chimeric protein. Mice wereinoculated with CT26 tumors on day 0. Once the tumors were palpable andat least 4 to 6 mm in diameter, mice were treated with two doses of 150μg of the mVSIG8-Fc-OX40L chimeric protein. Immunophenotyping wasperformed on various tissues collected from the mice 13 days after tumorimplantation.

FIG. 13A to FIG. 13C, show the results from the in vivo functionalassays. Immunoprofiling was performed on tumor-bearing mice treated withthe mVSIG8-Fc-OX40L chimeric protein. As shown in FIG. 13A, mice treatedwith the mVSIG8-Fc-OX40L chimeric protein exhibited higher percentagesof total CD4+ T cells in the spleen, peripheral lymph nodes, and tumorwhen compared to the untreated control mice. Within the spleen and thetumor, this increase in CD4+ T cell population was mostly due to anincrease in CD4+CD25− effector T cells, suggesting that activation ofnon-regulatory T cells is involved (FIG. 13B). The treated mice alsoexhibited a lower percentage of CD4+CD25+ regulatory T cells, suggestingthat regulatory T cells may be suppressed by the chimeric protein (FIG.13B).

The ability of the chimeric protein to stimulate the recognition oftumor antigens by CD8+ T cells was also analyzed. Specifically, FIG. 13Cshows tetramer staining analysis for determining the fraction of CD8+ Tcells that recognized the AH1 tumor antigen, which is natively expressedby CT26 tumors. Within the spleen, a higher proportion of CD8+ T cellswas found to recognize the AH1 tumor antigen in mice treated with themVSIG8-Fc-OX40L chimeric protein when compared to untreated controlmice. Notably, a much higher proportion of the AH1 tetramer-positiveCD8+ T cells was observed within tumor infiltrated lymphocytes (TIL) formice treated with the chimeric protein when compared to the untreatedcontrol mice.

Example 14: Characterization of the In Vivo Anti-Tumor Activities of theVSIG8-Fc-OX40L Chimeric Protein

The in vivo anti-tumor activity of the VSIG8-Fc-OX40L chimeric proteinwas analyzed using the CT26 mouse colorectal tumor models.

In one set of experiments, Balb/c mice were inoculated with CT26 tumorcells on day 0 and/or rechallenged with a second inoculation of CT26tumor cells at day 30. Following four days of tumor growth, when tumorsreached a diameter of 4 to 5 mm, mice were treated with either controlantibodies or 150 μg of the murine VSIG8-Fc-OX40L chimeric protein.Treatments were repeated on day seven. An analysis of the evolution oftumor size over 45 days after tumor inoculation was conducted.

As shown in FIG. 14A, the untreated mice developed significant tumorswhereas most of the mice treated with the mVSIG8-Fc-OX40L chimericprotein did not develop tumors of detectable size. FIG. 14B, shows theoverall survival percentage of mice through 50 days after tumorinoculation. All of the untreated mice died within 21 days after tumorinoculation, whereas mice treated the mVSIG8-Fc-OX40L chimeric proteinshowed a 100% survival rate at 30 days after tumor inoculation. At 50days after tumor inoculation, over 75% of the mVSIG8-Fc-OX40L-treatedmice remained alive. FIG. 14C summarizes the treatment outcomes for eachgroup. As shown in FIG. 14C, treatment with the chimeric protein alsoresulted in significantly higher tumor rejection than treatment with thecontrol antibodies. Notably, some mice exhibited prolonged tumorstabilization, consistent with an equilibrium effect between tumorgrowth and anti-tumor immunity. This period extended well beyond thelast time of chimeric protein treatment, and suggests that memoryimmunity may have been activated. Interestingly, some of the micewherein immune equilibrium was observed underwent delayed complete tumorrejections. More specifically, treatment with the chimeric proteinresulted in rejection of primary tumors as well as complete rejection oftumor cells administered during rechallenge, suggesting that memory Tcells may be involved.

The above data suggests that treatments with a VSIG8-Fc-OX40L chimericprotein creates an immune memory effect in vivo. Thus, the treatedanimal is able to later attack tumor cells and/or prevent development oftumors when rechallenged after an initial treatment with the chimericprotein.

Example 15: ELISA-Based Anti-Drug Antibody Assay of the MurineVSIG8-Fc-OX40L Chimeric Protein

An ELISA-based anti-drug antibody (ADA) assay was performed with themurine VSIG8-Fc-OX40L chimeric protein. High-binding ELISA plates werecoated either with animal serum (blue lines; starting undiluted (“neat”)and then dilutions of 1:2, 1:4, and 1:8) or recombinant murine Fc (blackline; starting at 5 μg/mL, and then dilutions of 1:2, 1:4, 1:8, 1:16,and 1:32), were probed with the mVSIG8-Fc-OX40L chimeric protein at 10μg/mL. Serum was collected from mice that rejected both primary andsecondary CT26 tumors, and were then challenged with a third dose of 150μg of the mVSIG8-Fc-OX40L chimeric protein via intraperitoneal (IP)injection on day 91. Serum was collected one week later on day 98. Thechimeric protein was detected using a goat-anti-mouse-OX40L antibody,followed by an anti-goat-HRP tertiary antibody. Absorbance values(OD₄₅₀) and the non-linear fit of each curve are shown in FIG. 15.

Example 16: Characterization of the Contribution of an Fc Domain in aLinker to Functionality of Chimeric Proteins

In this example, the contribution of an Fc domain in a linker tofunctionality of chimeric proteins of the present invention was assayed.Here, a PD1-Fc-OX40L was used as a model for Fc-containing chimericproteins. Thus, the data presented below is relevant to chimericproteins of the present invention.

In its native state, PD1 exists as monomer whereas OX40Ls tend todimerize due to electrostatic interactions between the OX40L domains; Fcdomains associate with each other via disulfide bonds. Together, severalinter-molecular interactions may contribute to the quaternary structureof PD1-Fc-OX40L. There are, at least, four potential configurations ofPD1-Fc-OX40L, with the chimeric protein existing as a monomer, a dimer,a trimer, or a hexamer. See, FIG. 16.

The existence of monomeric and dimeric configurations of the chimericprotein was tested by exposing chimeric proteins to reducing andnon-reducing conditions and then running the proteins on SDS-PAGE. Undernon-reducing conditions (Reduced: “−”), the chimeric protein migrated inSDS-PAGE at about 200 kDa. Here, Western blots were probed withantibodies directed against PD1, Fc, or OX40L in, respectively, theleft, middle, and right blots shown in FIG. 17. Since, the predictedmonomeric molecular weight of the chimeric protein is 57.6 kDa, the 200kDa species was expected to be, at least a dimer. However, under reducedconditions (Reduced: “+”), which reduces disulfide bonds (e.g., betweenFc domains), the chimeric protein migrated in SDS-PAGE at about 100 kDa.Since the 100 kDa species was heavier than expected, it was predictedthat the extra mass was due to glycosylation. Finally, chimeric proteinswere treated with Peptide-N-Glycosidase F (PNGaseF “+”) and run onSDS-PAGE under reduced conditions. Under these conditions, the chimericprotein migrated at about 57.6 kDa. These data suggest that the chimericprotein is glycosylated and exists naturally, at least, as a dimer; withdimerization likely due to disulfide bonding between Fc domains.

SDS-PAGE gel methods do not accurately predict the molecular weight forhighly charged and/or large molecular weight proteins. Thus, chimericproteins were next characterized using Size Exclusion Chromatography(SEC). Unlike SDS-PAGE, in which the negatively-charged SDS reducescharge-based interactions between peptides, SEC does not use detergentsor reducing agents. When the PD1-Fc-OX40L chimeric protein was run onSEC, none of the peaks were around 200 kDa. This suggests, thatnatively, the chimeric protein does not exist as a dimer. Instead, apeak having a size greater than 670 kDa was detected. See, FIG. 18. Thisand the prior data suggests that the PD1-Fc-OX40L chimeric proteinexists as a hexamer in its native state.

As shown above, when run on SDS-PAGE under non-reducing conditions orunder reducing conditions, SDS in the sample and/or running bufferconverts the hexameric PD1-Fc-OX40L chimeric protein into a predominantdimer or monomer, respectively, in the absence and presence of areducing agent. See, FIG. 19 (left gel). When run on native PAGE, whichlacks SDS, and in the absence of a reducing agent, the chimeric proteinexists as a hexamer. However, when run on native PAGE and in thepresence of a reducing agent (which reduces disulfide bonds) thechimeric protein migrated heavier than expected; as shown in FIG. 19(right gel, lane #2), with the chimeric protein failed to substantiallymigrate out of the loading well. This data suggests that the chimericprotein has oligomerized into a higher order protein. Thus, in chimericproteins, disulfide bonding appears to be important for controllinghigher-order oligomerization.

To further confirm this, chimeric proteins lacking an Fc domain wereconstructed, e.g., “PD1-No Fc-OX40L”. Such chimeric proteins will nothave the disulfide bonding which occurs between Fc domains in thechimeric proteins described previously. As shown in FIG. 20, whenchimeric proteins lacking Fc domains are run on native PAGE, none of theprotein substantially migrated out of its loading well (lane #1 to #4show increasing loading concentrations of PD1-No Fc-OX40L); again,suggesting that the “No Fc” chimeric proteins have formed aconcatemer-like complex comprising numerous proteins. Thus, omission ofthe Fc domain in a chimeric protein leads to formation of proteinaggregates. These data indicate that disulfide bonding, e.g., between Fcdomains on different chimeric proteins, stabilizes the chimeric proteinsand ensures that they each exist as a hexamer and not as a higher orderprotein/concatemer. In other words, the Fc domain surprisingly putsorder to chimeric protein complexes. Lane #1 to #4, respectively,include 2.5 μg, of PD1-No Fc-OX40L, 5 μg of PD1-No Fc-OX40L, 7.5 μg ofPD1-No Fc-OX40L, and 10 μg of PD1-No Fc-OX40L.

Shown in FIG. 21, is a model summarizing the above data and showing howa hexamer and concatemers form from chimeric proteins of the presentinvention. The exemplary chimeric protein (PD1-Fc-OX40L) naturally formsinto a hexamer (due to electrostatic interactions between the OX40Ldomains and dimerization by Fc domains). However, in the absence of thecontrolling effects of disulfide bonding between Fc domains, underreduced conditions for the PD1-Fc-OX40L protein and due to the absenceof Fc domains in the PD 1-No Fc-OX40L, these latter chimeric proteinsform concatemers.

Additionally, chimeric proteins were constructed in which the Fc domain(as described herein) was replaced with Ficolin (which lacks cysteineresidues necessary for disulfide bonding between chimeric proteins). Aswith the “No Fc” chimeric proteins and chimeric proteins comprising anFc and run on native PAGE and in the presence of a reducing agent (bothof which formed aggregates that do not migrate into a gel), chimericproteins comprising Ficolin appear to also form higher-order latticeswhich did not migrate into a gel. These data reinforce the conclusionthat disulfide binding is important for proper folding and function ofchimeric proteins of the present invention.

Finally, chimeric proteins were prepared using coiled Fc domains(CCDFc). Very little purified protein was delivered under functionalevaluation.

Accordingly, including an Fc domain in a linker of a chimeric protein(which is capable of forming disulfide bonds between chimeric proteins),helps avoid formation of insoluble and, likely, non-functional proteinconcatemers and/or aggregates.

Example 17: Production of Additional VSIG8-Containing Chimeric ProteinsComprising Extracellular Domains of Other Type II Proteins

In this example, additional chimeric proteins of the present inventionare described. Such additional chimeric proteins will be made similar tohow the VSIG8-Fc-OX40L chimeric proteins were made, e.g., as describedabove in the Detailed Description and in U.S. 62/464,002, the contentsof which are hereby incorporated by reference in its entirety.

These additional chimeric proteins will have the general formula: ECD1—Joining Linker 1—Fc Domain—Joining Linker 2—ECD 2, in which ECD 1 isthe extracellular domain of VSIG8 and ECD 2 is the extracellular domainof a type II protein, other than OX40L. Exemplary type II proteinsinclude 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A, and TRAIL. Thesechimeric proteins may lack one or both of the joining linkers. ExemplaryJoining Linker 1s, Fc Domains, and Joining Linker 2s are described abovein Table 1; modular linkers useful for forming chimeric proteins andcomprising specific Joining Linker 1s, Fc Domains, and Joining Linker 2sare shown in FIG. 22.

Alternately, the additional chimeric proteins will be fusion proteinshaving the general formula: N terminus—(a)—(b)—(c)—C terminus, in which(a) is VSIG8, (b) is a linker comprising at least a portion of a Fcdomain, and (c) is the extracellular domain of a type II protein otherthan OX40L. Exemplary type II proteins include 4-1BBL, CD30L, CD40L,FasL, GITRL, LIGHT, TL1A, and TRAIL. Exemplary linkers are describedabove in Table 1; modular linkers useful for forming chimeric proteinsand comprising specific Joining Linker 1s, Fc Domains, and JoiningLinker 2s are shown in FIG. 22.

The amino acid sequence for 4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT,TL1A, and TRAIL, respectively, comprises SEQ ID NO: 7, 9, 11, 13, 15,17, 21, and 23. The amino acid sequence for extracellular domain of4-1BBL, CD30L, CD40L, FasL, GITRL, LIGHT, TL1A, and TRAIL, respectively,comprises SEQ ID NO: 8, 10, 12, 14, 16, 18, 22, and 24. The amino acidsequence for VSIG8 comprises SEQ ID NO: 1 and the extracellular domainof VSIG8 comprises SEQ ID NO: 2. The chimeric proteins may comprise avariant of the above-mentioned sequences, e.g., at least about 95%identical to an above-mentioned sequence.

According, the present invention further includes the followingadditional chimeric proteins and methods using the additional chimericproteins (e.g., in treating a cancer and/or treating an inflammatorydisease): VSIG8-Fc-4-1BBL, VSIG8-Fc-CD30L, VSIG8-Fc-CD40L,VSIG8-Fc-FasL, VSIG8-Fc-GITRL, VSIG8-Fc-LIGHT, VSIG8-Fc-TL1A, andVSIG8-Fc-TRAIL.

The additional chimeric proteins will be characterized as describedabove for CSF1R-Fc-CD40L in Examples 1 to 15, albeit with reagents(e.g., binding partners, recombinant target cells, and cancer cell/tumortypes) that are specific to the additional chimeric proteins rather thanas needed for characterizing VSIG8-Fc-OX40L. Thus, using VSIG8-Fc-4-1BBLas an example, characterizations of VSIG8-Fc-4-1BBL akin to Example 2can be performed using anti-VSIG8, anti-Fc, and anti-4-1BBL antibodiesrather than the anti-VSIG8, anti-Fc, and anti-OX40L antibodies neededfor VSIG8-Fc-OX40L.

As with the VSIG8-Fc-OX40L chimeric proteins, the additional chimericproteins will be effective in treating a cancer and/or treating aninflammatory disease by blocking VSIG8 (which inhibits the transmissionof an immune inhibitory signal) and enhancing, increasing, and/orstimulating the transmission of an immune stimulatory signal viaactivating the receptor/ligand of one of 4-1BBL, CD30L, CD40L, FasL,GITRL, LIGHT, TL1A, and TRAIL. Moreover, the additional chimericproteins will be effective in treating a cancer and/or an inflammatorydisease yet without the toxicity resulting from treatments comprising aplurality of antibodies, e.g., a VISTA blocking antibody and an agonistantibody for the receptor/ligand of one of 4-1BBL, CD30L, CD40L, FasL,GITRL, LIGHT, TL1A, and TRAIL.

1.-63. (canceled)
 64. A heterologous chimeric protein, wherein theheterologous chimeric protein has a general structure of:N terminus−(a)−(b)−(c)−C terminus, wherein: (a) is a first domaincomprising an extracellular domain of V-set and immunoglobulindomain-containing protein 8 (VSIG8) that is capable of binding a VSIG8ligand, wherein the extracellular domain of VSIG8 comprises an aminoacid sequence that is at least 95% identical to the amino acid sequenceof SEQ ID NO: 2; (b) is a linker that comprises a hinge-CH2-CH3 Fcdomain derived from IgG4, and two or more joining linkers each joininglinker independently having an amino acid sequence selected from SEQ IDNOs: 28 to 74; wherein one joining linker is N terminal to thehinge-CH2-CH3 Fc domain and another joining linker is C terminal to thehinge-CH2-CH3 Fc domain; and (c) is a second domain comprising portionof 4-1BB ligand (4-1BBL) that binds a 4-1BBL receptor.
 65. Theheterologous chimeric protein of claim 64, wherein the first domaincomprises an amino acid sequence that is at least 97% identical to theamino acid sequence of SEQ ID NO:
 2. 66. The heterologous chimericprotein of claim 65, wherein the first domain comprises an amino acidsequence that is at least 99% identical to the amino acid sequence ofSEQ ID NO:
 2. 67. The heterologous chimeric protein of claim 64, whereinthe second domain comprises an amino acid sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8.68. The heterologous chimeric protein of claim 64, wherein the seconddomain comprises an amino acid sequence that is at least 95% identicalto the amino acid sequence of SEQ ID NO:
 8. 69. The heterologouschimeric protein of claim 68, wherein the second domain comprises anamino acid sequence that is at least 97% identical to the amino acidsequence of SEQ ID NO:
 8. 70. The heterologous chimeric protein of claim69, wherein the second domain comprises an amino acid sequence that isat least 99% identical to the amino acid sequence of SEQ ID NO:
 8. 71.The heterologous chimeric protein of claim 64, wherein the first domaincomprises an amino acid sequence that is at least 97% identical to theamino acid sequence of SEQ ID NO: 2; and the second domain comprises anamino acid sequence that is at least 97% identical to the amino acidsequence of SEQ ID NO:
 8. 72. The heterologous chimeric protein of claim64, wherein the linker comprises a sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, orSEQ ID NO:
 27. 73. The heterologous chimeric protein of claim 64,wherein the chimeric protein is expressed by a mammalian host cell as asecretable and functional single polypeptide chain.
 74. A nucleic acidencoding a polypeptide encoding the heterologous chimeric protein ofclaim
 64. 75. A host cell, comprising an expression vector comprisingthe nucleic acid of claim
 74. 76. A recombinant fusion proteincomprising: (a) a first domain comprising an extracellular domain ofV-set and immunoglobulin domain-containing protein 8 (VSIG8) that iscapable of binding a VSIG8 ligand, and comprising an amino acid sequencethat is at least 95% identical to the amino acid sequence of SEQ ID NO:2; (b) a linker that comprises a hinge-CH2-CH3 Fc domain, and two ormore joining linkers each joining linker independently having an aminoacid sequence selected from SEQ ID NOs: 28 to 74; wherein one joininglinker is N terminal to the hinge-CH2-CH3 Fc domain and another joininglinker is C terminal to the hinge-CH2-CH3 Fc domain; and (c) a seconddomain comprising an extracellular domain of 4-1BB ligand (4-1BBL) thatbinds a 4- 1BBL receptor and comprises an amino acid sequence that is atleast 95% identical to the amino acid sequence of SEQ ID NO:
 8. 77. Therecombinant fusion protein of claim 76, wherein the linker comprises asequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO:
 27. 78. The recombinantfusion protein of claim 77, wherein the first domain comprises an aminoacid sequence that is at least 98% identical to the amino acid sequenceof SEQ ID NO: 2; and/or the second domain comprises an amino acidsequence that is at least 98% identical to the amino acid sequence ofSEQ ID NO:
 8. 79. The recombinant fusion protein of claim 78, whereinthe first domain comprises an amino acid sequence that is at least 98%identical to the amino acid sequence of SEQ ID NO: 2; and the seconddomain comprises an amino acid sequence that is at least 98% identicalto the amino acid sequence of SEQ ID NO:
 8. 80. A nucleic acid encodinga polypeptide encoding the recombinant fusion protein of claim
 76. 81. Apharmaceutical composition, comprising the heterologous chimeric proteinof claim
 64. 82. A pharmaceutical composition, comprising therecombinant fusion protein of claim
 76. 83. A method for treating canceror an inflammatory disease comprising administering a pharmaceuticalcomposition to a subject in need thereof, the pharmaceutical compositioncomprising a heterologous chimeric protein, wherein the heterologouschimeric protein has a general structure of:N terminus−(a)−(b)−(c)−C terminus, wherein: (a) is a first domaincomprising an extracellular domain of V-set and immunoglobulindomain-containing protein 8 (VSIG8) that is capable of binding a VSIG8ligand, wherein the extracellular domain of VSIG8 comprises an aminoacid sequence that is at least 95% identical to the amino acid sequenceof SEQ ID NO: 2; (b) is a linker that comprises a hinge-CH2-CH3 Fcdomain derived from IgG4, and two or more joining linkers each joininglinker independently having an amino acid sequence selected from SEQ IDNOs: 28 to 74; wherein one joining linker is N terminal to thehinge-CH2-CH3 Fc domain and another joining linker is C terminal to thehinge-CH2-CH3 Fc domain; and (c) is a second domain comprising portionof 4-1BB ligand (4-1BBL) that binds a 4-1BBL receptor.